Therapy of sarcoidosis

ABSTRACT

Sarcoidosis is associated with CD4 +   T lymphocytes which express the T cell receptor V.sub.α 2.3 chain. Thus, a method for diagnosing sarcoidosis is provided which comprises contacting cells of a subject with a first monoclonal antibody, or an antigen-binding fragment or derivative, specific for an epitope of the variable region of the T cell receptor V.sub.α 2.3 chain and detecting the binding of the antibody. Also provided is a method for treating sarcoidosis in which a monoclonal antibody, or an antigen-binding fragment or derivative thereof, specific for an epitope of the variable region of the T cell receptor V.sub.α 2.3 chain is administered. Sarcoidosis is also treated by administering a therapeutically effective amount of a protein or a peptide comprising an amino acid sequence of the variable region of the T cell receptor V.sub.α 2.3 chain, or a functional derivative of the protein or peptide, or an antisense oligonucleotide which is complementary to the T cell receptor V.sub.α 2.3 mRNA.

1. INTRODUCTION

The present invention in the fields of immunology and medicine relatesto methods for diagnosing and treating sarcoidosis based on the presencein the lungs of sarcoidosis patients of T lymphocytes expressing the V₆₀2.3 variant of the T cell receptor a chain. Monoclonal antibodiesspecific for an epitope of the variable region of the T cell receptorV₆₀ 2.3 chain, or epitope-binding fragments or derivatives of theantibody, are useful in diagnostic and therapeutic methods.

2. BACKGROUND OF THE INVENTION 2.1. Sarcoidosis

Sarcoidosis is a chronic inflammatory disorder with unknown etiology,characterized by non-caseating granulomas in affected organs, inparticular, the lungs, lymph nodes, skin and eyes. The disorder istypically accompanied by nonspecific depression of cell-mediated as wellas humoral immune responsiveness, and by polyclonalhypergamma-globulinemia (Siltzbach, L. E., Amer. Rev. Resp. Dis. 97:1-8(1968); Roberts, C. R. et al., Ann. Intern. Med. 94:73 (1981)). At least90% of the patients with this multisystem disease have pulmonarymanifestations characterized by chronic inflammation, granulomaformation and some cases of pulmonary fibrosis. These processes affectthe alveoli, airways and blood vessels resulting in an impairment ofnormal gas exchange. The inflammatory process precedes the othersymptoms of sarcoidosis.

CD4⁺ T helper (Th) cells are believed to play a central role in thepathogenesis of sarcoidosis. Such activated cells accumulate in thealveolar space, spontaneously release IL2 and proliferate at high ratesin vitro and express HLA-DR, a marker of T cell activation (Hunninghake,G. et al., N. Engl. J. Med. 305:429 (1981)). The T cells in the lungwhich spontaneously release IL2 are primarily of the CD4⁺ HLA-DR⁺ class(Saltini, C. et al., J. Clin. Invest. 77:1962-1970 (1986)). The releaseof cytokines results in modulation of granuloma formation and polyclonalactivation of B cells to secrete immunoglobulin (Hunninghake et al.,supra). A subset of Th cells identified by a mAb designated 5/9, whichdetects activated T cells, was shown to predominate in the lungs ofsarcoidosis patients and was responsible for the release of lymphokinesand the polyclonal B cell activation (Rossi, G. A. et al., Am. Rev.Respir. Dis. 133:1086-1090 (1986)). In sarcoidosis patients withhigh-intensity alveolitis, T lymphocytes from lung (but not those fromperipheral blood) spontaneously release IL2 in vitro and replicate at ahigh rate (Pinkston, P. et al., N. Engl. J. Med. 308:793 (1983)).

2.2. The T Cell Antigen Receptor

T lymphocytes recognize and interact with antigens by means of acell-surface molecular complex known as the T cell antigen receptor(TCR) complex. The TCR is a clone-specific heterodimeric protein on Tcells, which recognizes its "target" antigen in association with a majorhistocompatibility complex (MHC)-encoded glycoprotein on the surface ofantigen presenting cells (APC). CD4⁺ T cells recognize predominantlyantigen associated with MHC class II molecules whereas CD8⁺ T cellsrecognize antigen associated with MHC class I molecules. The TCR isnoncovalently associated with the CD3 complex of molecules.Approximately 90% of peripheral blood T cells express a TCR which is aheterodimer of an α and a β chain. A small percentage of T cells expressa TCR consisting of a heterodimer comprising a γ and a δ polypeptidechain. (See, for example, Davis et al., 1988, Nature 334:395-402;Marrack et al., 1986, Sci. Amer. 254:36; Meuer et al., 1984, Ann. Rev.Immunol. 2:23-50; Brenner et al., 1986, Nature 322:145-159; Krangel etal., 1987, Science 237:1051-1055; Hata et al., 1987, Science238:678-682; Hochstenbach et al., 1988, J. Exp. Med. 168:761-776).

In a given T cell or clone of T cells, each TCR chain is a uniquecombination of domains designated variable (V), (diversity (D),! joining(J), and constant (C) (Siu et al., 1984, Cell 37:393; Yanagi et al.,1985, Proc. Natl. Acad. Sci. USA 82:3430). Hypervariable regions havealso been identified (Patten et al., 1984, Nature 312:40; Becker et al.,1985, Nature 317:430). The V domains of the TCR α and β chains arecreated through rearrangement of each of 60 V and 75 J and of one eachof 70 V, 2 D and 13 J gene segments, respectively (Ferradini, L. et al.,Eur. J. Immunol. 21:935 (1991); Roman-Roman, S. et al., Eur. J. Immunol.21:927 (1991)). Adding further junctional and N-linked diversity, andmultiple reading frames for D segments, the calculated variability ofthe α/β TCR is in the order of 10¹⁵ (Davis, M. et al., Nature 334:395(1988)). In each T cell clone, the combination of V, D and J domains ofboth the α and the β chains or of both the δ and γ chains participatesin antigen recognition in a manner which is uniquely characteristic ofthat T cell clone and defines a unique "binding site," also known as theidiotype, of the T cell clone. In contrast, the TCR C domain does notparticipate in antigen binding.

2.3. T Cell Receptor Expression in Sarcoidosis

Certain diseases, in particular those with autoimmune etiologies, areassociated with the increase in frequencies of T lymphocytes expressinga particular α or β TCR V region gene (Hafler, D. A. et al., J. Exp.Med. 167:1313 (1988); Mantegazza, R. et al., Autoimmunity 3:431 (1990)).Such limited or preferential usage of specific TCRs has recently beenobserved in sarcoidosis (Moller, D. et al., J. Clin. Invest. 82:1183(1988); Balbi, B. et al., J. Clin. Invest. 85:1353 (1990); Tamura, N. etal., J. Exp. Med. 172:169 (1990)).

Moller et al., (supra) found that, in a subgroup of sarcoidosispatients, T cells obtained from lungs showed an increase in thepercentage of CD4⁺ cells expressing the TCR V.sub.β 8 chain. Incontrast, in the blood of the same patients, there was an increasedprevalence of V₆₂ 8⁺ cells in the CD⁺ T cell population. The authorsconcluded that a selective accumulation of T cells expressing aparticular TCR β chain occurred in this disease.

Balbi et al., (supra) disclosed a marked elevation of CD3⁺ TCRγδ⁺ Tcells in the lungs and blood of sarcoidosis patients, with the majorityof the γδ⁺ T cells expressing a particular γ chain V region, V.sub.γ 9.

Tamura et al., (supra) further analyzed TCR Vγ (and Vδ) chain usage insarcoidosis patients by sequencing junctional (N) regions of the commonhuman V.sub.γ 9 gene segments. They disclosed a broad diversity ofV.sub.γ 9 junctional sequences in normals, but a strikingover-representation of specific junctional region sequences in asubgroup of sarcoidosis patients. The authors speculated that this typeof TCR usage represents a response to specific antigenic stimuli andthat the γ/δ T cells might play a specific role in granuloma formation.

2.4. Antisense Oligonucleotides as Therapeutic Agents

Antisense oligonucleotides are thought to inhibit gene expression byblocking the processing and translation of the sense mRNA or bydisrupting interactions with sequence-specific RNA binding proteins. Forexample, a plasmid having a promoter which directs transcription of RNAcomplementary to normal thymidine kinase (TK) mRNA substantially reducesexpression of TK from a normal plasmid with which it is cotransfectedinto a cell (Izant et al., Cell 36:1007 (1984).

The constitutive expression of antisense RNA in cells can inhibit theexpression of numerous genes in mammals and plants, and the listcontinually grows (Hambor, J. E. et al., J. Exp. Med. 168:1237-1245(1988); Holt, J. T. et al., Proc. Natl. Acad. Sci. USA 83:4794-4798(1986); Izant, J. G. et al., Cell 36:1007-1015 (1984); Izant, J. G., etal., Science 229:345-352 (1985); De Benedetti, A. et al., Proc. Nat..Acad. Sci. 84:658-662 (1987)).

Antisense effects may be due to blockage of translation or prevention ofsplicing, both of which have been observed in vitro. Interference withsplicing also allows the use of intron sequences (Munroe, S. H., EMBO.J. 7:2523-2532 (1988) which should be less conserved and thereforeresult in greater species specificity of inhibition.

Antisense technology has been applied successfully to primary human Tlymphocytes to inhibit the replication of a virus, HTLV-I (Ruden et al.,J. Virol. 63:677-682 (1989)) by engineering the cells to express anantisense RNA to parts of the viral genome. For example,antisense-encoding DNAs operably linked to the cytomegalovirus immediateearly promoter expressed antisense RNA and exerted an inhibitory effecton cell proliferation.

3. SUMMARY OF THE INVENTION

The present invention relates to methods for diagnosing and treatingsarcoidosis using a binding partner specific for the variable region ofthe TCR V₆₀ 2.3 chain, preferably antibodies specific for epitopes ofthe TCR V₆₀ 2.3 chain variable region. These approaches are based on thepresent inventors' discovery that a particular subset of CD4⁺lymphocytes which express a TCR recognized by an antibody specific forthe TCR V.sub.α 2.3 chain is preferentially compartmentalized in thelungs of sarcoidosis patients. In particular, levels of V.sub.α2.3-positive cells were increased among CD4⁺ T cells in bronchoalveolarlavage (BAL) relative to CD⁺ peripheral blood lymphocyte (PBL)populations.

Thus, the present invention is directed to a method for diagnosingsarcoidosis in a patient suspected of having sarcoidosis, comprisingdetecting an increase in the number of cells expressing a T cell antigenreceptor V.sub.α 2.3 chain in a sample from the patient, relative to thenumber of cells in a comparable sample obtained from a healthy subjector relative to the number in a baseline sample.

The present invention further provides a method for diagnosingsarcoidosis in a patient suspected of having sarcoidosis, comprising:

(a) determining the ratio of the number of cells expressing a T cellantigen receptor V.sub.α 2.3 chain to the number of a second group ofcells, such as total lymphocytes, total T cells or CD4-positive T cells,in a sample from the lungs or from bronchoalveolar lavage fluid obtainedfrom the patient; and

(b) comparing the ratio determined in step (a) with the ratio of thenumber of cells expressing a T cell receptor V.sub.α 2.3 chain to thenumber of cells of the second group in a sample from the lungs or frombronchoalveolar lavage fluid obtained from a healthy subject, or in theblood of the patient or the healthy subject, or with a baseline sampleratio;

wherein an increased ratio of the number of cells expressing the V.sub.α2.3 chain in the lungs or bronchoalveolar lavage fluid of the patientindicates the presence of sarcoidosis.

Preferably, in the above method, the detecting is performed on cells inbronchoalveolar lavage fluid obtained from the patient, and thecomparing is with the ratio of cells in the peripheral blood of thepatient.

Preferably, the determining step above comprises:

(i) contacting cells of the patient with a first binding partner whichbinds specifically to the variable region of the T cell receptor V.sub.α2.3 chain; and

(ii) detecting the specific binding of the binding partner to the cells.

The first binding partner above is preferably an antibody, mostpreferably a monoclonal antibody, specific for an epitope of thevariable region of the T cell receptor V.sub.α 2.3 chain, or anepitope-binding fragment or derivative of the antibody. A most preferredmonoclonal antibody is has binding characteristics of F1, as produced bythe hybridoma deposited with the ATCC and assigned accession number HB11176.

Another embodiment of the above method further comprises, before, duringor after step (a), determining the number of CD4-positive cells in thelungs or bronchoalveolar lavage fluid, such as by detecting the bindingto the cells of a second binding partner specific for the CD4 molecule,preferably a second monoclonal antibody specific for an epitope of theCD4 molecule, or an epitope-binding fragment or derivative of the secondantibody.

The contacting in the above methods may be in vitro or in vivo. Thesample may be a histological specimen, such as lung tissue.

The present invention also provides a method for diagnosing sarcoidosisin a patient suspected of having sarcoidosis, comprising:

(a) determining the number of T lymphocytes expressing the variableregion of the T cell receptor V.sub.α 2.3 chain in a sample containing Tlymphocytes from the lungs or bronchoalveolar lavage fluid of thepatient;

(b) determining the number of CD4-positive T lymphocytes in the sample;

(c) determining the percentage of CD4-positive cells which express thevariable region of the T cell receptor V.sub.α 2.3 chain in the sample;and

(d) comparing the percentage determined in step (c) with the percentageof CD4-positive cells which express the variable region of the T cellantigen receptor V.sub.α 2.3 chain in a sample containing T lymphocytesfrom the lungs or bronchoalveolar lavage fluid of a healthy subject orfrom the peripheral blood of the patient or the healthy subject, or abaseline sample percentage,

wherein an increased percentage of CD4-positive cells expressing theV.sub.α 2.3 chain in the lungs or bronchoalveolar lavage fluid of thepatient indicates the presence of sarcoidosis.

In the above method,

(i) the number of T lymphocytes expressing the variable region of the Tcell receptor V.sub.α 2.3 chain is preferably determined by contactingthe cells with a first monoclonal antibody specific for the variableregion of the T cell receptor V.sub.α 2.3 chain, or an epitope-bindingfragment or derivative of the monoclonal antibody, and detecting theimmunospecific binding of the first antibody, fragment or derivative tothe T lymphocytes; and

(ii) the number of CD4-positive T lymphocytes is preferably determinedby contacting the cells with a second monoclonal antibody specific forthe CD4 molecule, or an epitope-binding fragment or derivative of thesecond antibody, and detecting the binding of the second antibody,fragment or derivative to the T lymphocytes.

A preferred first monoclonal antibody has binding characteristics of F1,as produced by the hybridoma deposited with the ATCC and assignedaccession number HB 11176.

Also provided is a method for diagnosing sarcoidosis in a subjectsuspected of having sarcoidosis, comprising detecting in a nucleic acidpreparation derived from a T lymphocyte-containing sample from thesubject the presence of rearranged nucleic acid sequence, mRNA or DNA,encoding the variable region of a T cell receptor V.sub.α 2.3 chain.

The present invention is also directed to methods for treatingsarcoidosis in a subject. In a preferred embodiment, the methodcomprises administering to the subject a therapeutically effectiveamount of a binding partner of the T cell receptor V.sub.α 2.3 chain. Apreferred binding partner is an antibody, preferably a monoclonalantibody specific for an epitope of the variable region of the T cellreceptor V.sub.α 2.3 chain, or an epitope-binding fragment or derivativeof the monoclonal antibody. A preferred monoclonal antibody has bindingcharacteristics of F1, as produced by the hybridoma deposited with theATCC and assigned accession number HB 11176. The binding partner,preferably an antibody, fragment, or derivative thereof, may be linkedto a pharmacologic agent.

Also provided is a method of treating sarcoidosis in a subject,comprising administering to the subject a therapeutically effectiveamount of a protein or a peptide having at least about 15 amino acidscomprising an amino acid sequence of the variable region of the T cellreceptor V.sub.α 2.3 chain (FIG. 4). Preferably the protein or peptideis selected from the group consisting of peptides having the amino acidsequence:

(a) MMISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNIRPGNTPLVFGKGTRLSVIPNI (SEQ ID NO:2);

(b) KEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNIRPGNTPL VFGKGTRLSVIPNI (SEQ IDNO:3);

(c) KEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVN (SEQ ID NO:4);

(d) IRPGNTPLVFGKGTRLSVIPNI (SEQ ID NO:5);

(e) KEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFW (SEQ ID NO:6);

(f) YRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSD (SEQ ID NO:7); and

(g) SATYLCVVNIRPGNTPLVFGKGTRLSVIPNI (SEQ ID NO:8).

The peptide may preferably have between about 15 and 32 amino acids, andis preferably selected from the group consisting of the peptides havingthe amino acid sequence:

(a) KEVEQDPGPFNVPEGATVAFN (SEQ ID NO:9);

(b) CTYSNSASQSFFWYRQD (SEQ ID NO:10);

(c) CRKEPKLLMSVYSSGN (SEQ ID NO:11);

(d) EDGRFTAQLNRASQYISLLIRDSKLSDSATYL (SEQ ID NO:12); and

(e) CVVNIRPGNTPLVFGKGTRLSVIPNI (SEQ ID NO:13).

The above protein or peptide may be linked to a pharmacologic agent.Also provided are peptides or proteins comprising the foregoingsequences.

The present invention also includes a therapeutic composition useful forthe treatment of sarcoidosis, comprising:

(a) an effective amount of a T cell receptor V.sub.α 2.3 chain proteinor peptide, as above; and

(b) a suitable pharmaceutical carrier.

Another embodiment of a therapeutic composition useful for the treatmentof sarcoidosis comprises:

(a) an effective amount of a monoclonal antibody specific for an epitopeof the variable region of the T cell receptor V.sub.α 2.3 chain, or anantigen-binding fragment or derivative of the monoclonal antibody; and

(b) a suitable pharmaceutical carrier.

A preferred mAb is the F1 mAb

Also provided is an isolated antisense oligonucleotide consisting of atleast about fifteen nucleotides and comprising a sequence complementaryto at least a portion of an RNA transcript of the human TCR V.sub.α 2.3gene, which oligonucleotide is hybridizable to the RNA transcript and iscapable of interfering with expression of the V.sub.α 2.3 gene. Apharmaceutical composition comprises the above oligonucleotide and apharmaceutically acceptable carrier.

The present invention provides a composition comprising atherapeutically effective amount of a mAb specific for an epitope of thevariable region of the T cell receptor V.sub.α 2.3 chain, preferably theF1 mAb, or an antigen-binding fragment or derivative of the mAb for usein treating sarcoidosis.

Also provided is the use of a composition comprising a therapeuticallyeffective amount of a mAb specific for an epitope of the variable regionof the T cell receptor V.sub.α 2.3 chain, preferably the F1 mAb, or anantigen-binding fragment or derivative of the mAb, for the manufactureof a medicament in the treatment of sarcoidosis.

In another embodiment is provided a composition comprising atherapeutically effective amount of a protein or a peptide having atleast about 15 amino acids comprising an amino acid sequence of thevariable region of the T cell receptor V.sub.α 2.3 chain for use intreating sarcoidosis.

Also provided is the use of a composition comprising a therapeuticallyeffective amount of a protein or a peptide having at least about 15amino acids comprising an amino acid sequence of the variable region ofthe T cell receptor Va.sub.α2.3 chain for the manufacture of amedicament in the treatment of sarcoidosis.

4. DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the percentage of V.sub.α 2.3⁺ CD4⁺ T cells inPBL (□) and BAL (▪) from 11 patients with sarcoidosis (panel A) and fromfour controls (panel B).

FIG. 2 is a flow cytometry profile from a double-staining experimentwith lymphocyte gated BAL cells from patient no. 10. The ordinaterepresents cells stained with PE-conjugated (fluorescence two) anti-CD4.The abscissa represents cells detected by FITC-labeled (fluorescenceone): (a) normal mouse serum (NMS) (b) V.sub.α 2.3, and (c) OKT3. TheCD4⁺ cells positively stained with the FITC-labelled mAb were (a) 0.2%,(b) 31.9% and (c) 99.8%.

FIG. 3 is a diagram showing percentage of V.sub.α 2.3⁺ CD4⁺ cells inBAL(abscissa) and PBL (ordinate) in patient nos. 1 (×), 6 (▪), 7 () and10 (+) sampled for the first (a) and the second (b) analysis.

FIG. 4 shows the nucleotide sequence SEQ ID NO:1! of the entire variableregion of the TCR V.sub.α 2.3 gene, including the leader, V and Jregions.

FIG. 5 shows the amino acid sequence SEQ ID NO:2! of the entire variableregion of the TCR V.sub.α 2.3 protein chain. The boundaries of theleader, V and J regions are shown.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for diagnosisand therapy of sarcoidosis. The present inventors have discovered thatsarcoidosis patients show selective compartmentalization to the lung ofV.sub.α 2.3⁺ CD⁺ T cells. As many as about one third of the accumulatedCD4⁺ T cells in bronchoalveolar lavage (BAL) of sarcoidosis patientsspecifically expressed V.sub.α 2.3 chains. This restricted V gene usagesuggested the presence of an antigen which specifically stimulatedpulmonary V.sub.α 2.3⁺ CD4⁺ in sarcoidosis. In contrast, no significantcompartmentalization of such T cells occurs in healthy subjects.

The present inventors further have discovered a striking correlationbetween the accumulation of V.sub.α 2.3⁺ CD4⁺ T cells in the lung andthe HLA-DR3(w17), DQw2 haplotype. Thus, the present inventors havediscovered in a subgroup of sarcoidosis patients, two of the structuresin the specific trimolecular complex comprising the TCR, the MHCmolecule and the antigen. According to the present invention, theV.sub.α 2.3⁺ CD4⁺ T cells which selectively accumulate in the lung ofsarcoidosis patients are considered to play a pathophysiologic role inthe disease process by directly or indirectly mediating the sarcoidosisdisease.

Furthermore, a correlation between the localized V.sub.α 2.3⁺ CD4⁺ Tcells and the time course of the disease discovered by the presentinventors strengthens the association with the disease. Thus,enumeration of T cells expressing the TCR V.sub.α 2.3 in various cellpopulations is useful in diagnosing or predicting the course or responseto therapy of sarcoidosis.

Thus, the present invention provides diagnostic and therapeuticapproaches and agents for sarcoidosis based on the selective presence ofa select T cell subpopulation bearing the V.sub.α 2.3 variant of theTCRα chain. These approaches and reagents are described more fullybelow.

5.1. Antibodies

Antibodies as well as fragments, derivatives, or analogues thereof,specific for an epitope of the V.sub.α 2.3 region of a human TCR α chainmay be utilized in the diagnosis and therapy of sarcoidosis.

The term "antibody" is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), and chimeric antibodies (see below).Preferred antibodies are mAbs, which may be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, and any subclass or isotype thereof.Preferred antibodies for therapeutic use include antibodies of the IgG2aor IgG2b isotype (Rashid et al., 1992, J. Immunol. 148: 1382-1388).

The term "antibody" is also meant to include both intact molecules aswell as fragments thereof which bind the antigen, such as, for example,F(ab')₂, Fab', Fab and Fv. These fragments lack the Fc fragment of anintact antibody molecule, clear more rapidly from the circulation, andmay have less non-specific tissue binding than an intact antibody (Wahlet al., 1983, J. Nucl. Med. 24:316-325), properties which may bedesirable for particular therapeutic or diagnostic utilities. It will beappreciated that these antigen-binding or epitope-binding fragments ofthe antibodies useful in the present invention may be used for thedetection and quantitation of TCR proteins or peptides, or cellsexpressing the TCR proteins, as disclosed herein for intact antibodymolecules. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab')₂ fragments) or by reducing the disulfidebridges.

The mAbs of the invention are reactive with a variable region of theV.sub.α 2.3 variant of the α a chain of the TCR. In a specificembodiment, mAb F1 as deposited with the ATCC and assigned accessionnumber HB 11176 is used. The V.sub.α 2.3-specific mAbs of the presentinvention enables the analysis of the expression of the V.sub.α 2.3 genein a biological sample.

Various chemical or biochemical derivatives of the antibodies orantibody fragments of the present invention can also be produced usingknown methods. One type of derivative which is diagnostically useful isan immunoconjugate comprising an antibody molecule, or anantigen-binding fragment thereof, to which is conjugated a detectablelabel such as a radioisotope, a fluorescent dye or another tracermolecule. A therapeutically useful immunoconjugate comprises an antibodymolecule, or an antigen-binding fragment thereof, conjugated to atherapeutically useful molecule such as a cytotoxic drug or a toxicprotein (see, for review: Dillman, R. O., Ann. Int. Med. 111:592-603(1989)). Such antibody derivatives are discussed in more detail below.

The antibody, fragment or derivative useful in the present invention,may be prepared by using any of a number of techniques well-known in theart. For producing a mAb, any method which provides for the productionof antibody molecules by continuous cell lines in culture may be used.These methods include, but are not limited to,the hybridoma techniqueoriginally described by Kohler and Milstein, (1975, Nature 256:495-497),and the more recent human B cell hybridoma technique (Kozbor et al.,1983, Immunol. Today 4:72), EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96), and trioma techniques. A hybridoma of rodent origin producingthe mAbs of this invention may be cultivated in vitro or in vivo. For anoverview of antibody production methods, see: Hartlow, E. et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988.

In one embodiment, the antibody of the present invention is a human mAb.Human mabs may be made by any of a number of techniques known in the art(e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312;Kozbor et al., supra; Olsson et al., 1982, Meth. Enzymol. 92:3-16).

In another embodiment, the antibody is a chimeric antibody, preferably amouse-human chimeric antibody, wherein the heavy and light chainvariable regions are derived from a murine mAb and the constant regionsare of human origin. The chimeric antibodies of this invention have boththe TCR-recognizing specificity of the mouse Mab and the biologicalproperties of human antibodies, which include resistance to clearance inthe human and lower immunogenicity for humans, allowing multipletreatments. Methods for producing chimeric antibody molecules aredisclosed, for example, in Gorman et al., PCT Publication WO 92/06193(Apr. 16, 1992); Cabilly et al., U.S. Pat. No. 4,816,567 (Mar. 28, 1989)and EPO Publication EP125023 (Nov. 14, 1984); Taniguchi et al., EPOPublication EP171496 (Feb. 19, 1986); Morrison et al., EPO PublicationEP173494 (Mar. 5, 1986); Neuberger et al., PCT Publication WO 86/01533(Mar. 13, 1986); Kudo et al., EPO Publication EP184187 (Jun. 11, 1986);Robinson et al., PCT Publication WO 87/02671 (May 7, 1987); Boulianne etal., Nature 312:643-646 (1984); Morrison, Science 229:1202-1207 (1985);Neuberger et al., Nature 314:268-270 (1985); Takeda et al., Nature314:452-454 (1985); Oi et al., BioTechniques 4:214 (1986); and Liu etal., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987).

For human therapeutic purposes, mAbs or chimeric antibodies can be"humanized" by producing human constant region chimeras, where evenparts of the variable regions, in particular the conserved or frameworkregions of the antigen-binding domain, are of human origin, and only thehypervariable regions are non-human. See for example, Winter, UK PatentPublication GB 2188638A (Oct. 7, 1987); Harris et al., PCT PublicationWO 92/04381 (Mar. 19, 1992); Gorman et al., supra); Riechmann et al.,1988, Nature 332:323-327.

In yet another further embodiment, the antibody is a single chainantibody formed by linking the heavy and light chain fragment of the Fvregion via an amino acid bridge, resulting in a single chain polypeptide(Bird, 1988, Science 242:423-426; Huston et: al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883: and Ward et al., 1989, Nature 340:544-546).

Antibody molecules or fragments may be purified by known techniques,e.g., immunoabsorption or immunoaffinity chromatography, for example,using Staphylococcal protein A, or chromatographic methods such as HPLC(high performance liquid chromatography), or a combination thereof, etc.In a preferred method, the anti- V.sub.α 2.3 mAb, preferably F1(produced by the hybridoma line deposited in the ATCC under accessionnumber #HB 11176), is purified from culture supernatant or ascitesfluid.

Once antibodies of the desired specificity are generated, they may beused to identify and select other antibodies having the same orcross-reactive epitope specificity. For example, a new antibody istested by measuring its ability to inhibit the binding of an antibody ofknown specificity to its epitope. Various competitive binding assaysknown in the art can be used.

The isotype of the antibody can be selected during hybridoma productionor by appropriate recombinant methods well-known in the art to achieve adesired effector function mediated by the Fc portion of theimmunoglobulin heavy chain. For example, certain isotypes, such asIgG2a, have superior activity in antibody-dependent cellularcytotoxicity. Likewise, certain isotypes, such as IgG2a, are morereadily eliminated from the circulation through Fc receptors on cells ofthe reticuloendothelial system and are therefore more efficient atremoving an undesired antigen or target cell from sites of activedisease (Rashid et al., supra). Accordingly, depending on the intendeduse, a particular antibody isotype may be preferable to others, as canbe readily ascertained by one of ordinary skill in the art without undueexperimentation.

As used herein, an antibody reactive with the "V region" of the TCRshall be construed to be an antibody reactive with an epitope of the Vregion, a combination epitope of the V region, or a combination epitopeof the V-D or V-D-J regions. An antibody reactive with a V region of aTCR may recognize an idiotypic determinant, a clonotypic determinant,or, preferably, may recognize a minor framework region expressed by adiscrete subset of T lymphocytes. An "anti-clonotypic" antibody reactsonly with a determinant ("clonotypic determinant) of a particular cloneof T cells, generally that clone against which it was raised (Acuto etal., Cell 34:717-726 (1988); Meuer et al. Proc. Natl. Acad. Sci.81:1509-1513 (1984); Meuer et al., Ann. Rev. Immunol. 2:23-50 (1984)).Anti-clonotypic antibodies are also referred to as anti-idiotypicantibodies. "Minor framework region" refers to a region of the TCR whichis not shared by all TCR molecules, but is also not unique to aparticular T cell clone. Preferred anti-TCR a mAbs recognize members ofthe V.sub.α 2 family, most preferably, V.sub.α 2.3. Preferably, such anantibody is reactive with a unique epitope on a V.sub.α 2.3 variableregion of the α chain of the TCR.

5.2. Diagnostic Use of Antibodies

The antibodies and fragments described herein are useful for diagnosticor research purposes in various immunoassays well-known in the art. Theantibodies, or fragments of antibodies, useful according to the presentinvention may be used to quantitatively or qualitatively detect thepresence of cells which express the TCR V.sub.α 2.3 gene, or to measurethe levels of TCR V.sub.α 2.3 protein present in a sample. This can beaccomplished by immunofluorescence techniques employing a fluorescentlylabeled antibody (see below) coupled with light microscopic, flowcytometric, or fluorimetric detection.

The antibodies (or fragments or derivatives thereof) useful in thepresent invention may be employed histologically, as inimmunohistochemical staining, immunofluorescence or immunoelectronmicroscopy, for in situ detection of the TCR molecule.

One way of measuring the reactivity of a TCR epitope with a specificantibody of the present invention is by enzyme immunoassay (EIA) such asan enzyme-linked immunosorbent assay (ELISA) (Voller, A. et al., J.Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol.73:482-523 (1981); Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, BocaRaton, Fla., 1980). The enzyme, either conjugated to the antibody or toa binding partner for the antibody, when later exposed to an appropriatesubstrate, will react with the substrate in such a manner as to producea chemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means.

A preferred method of enumerating total TCR V.sub.α 2.3 chain isperformed using detergent treated whole blood samples or isolated T cellor CD4⁺ T cell populations. In particular V.sub.α 2.3-bearing subset maybe detected from a sample by adding the cells (or whole blood) to wellsof a 96 well microplate previously coated with 5 μg/ml of coatingantibody. Coating antibody is either a negative control or an anti-majorframework antibody to detect total a chain or a TCR V region specificmAb such F1 to detect a the V.sub.α 2.3 subset. An enzyme conjugatedantibody, which recognizes a different epitope, for example, an epitopeof the α or β a chain C region is used as a detection antibody. Theassay format is described in Rittershaus C. W., PCT Publication WO92/08981 (May 29, 1992).

Detection of the TCR V.sub.α 2.3 protein or cells expressing the proteinmay be accomplished using any of a variety of other immunoassays. Forexample it is possible to detect antibody binding to TCR V regionthrough the use of a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986, pp.1-5, 46-49 and 68-78).

It is also possible to label the antibody in which binding is measuredusing radioactive, fluorescent, chemiluminescent or bioluminescentconjugated antibodies.

A variety of immunoassay formats is available, for either EIA or RIAsystems. For example, assays may be competitive or non-competitive. Twosite or sandwich assays may be used, either "forward", "simultaneous" or"reverse" assays, which are well-known in the art.

Additional types of immunoassays include precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement fixation assays, immunoradiometric assays, protein Aimmunoassays, and immunoelectrophoresis assays.

Binding of the antibody, or fragment or derivative thereof to the TCRepitope for which it is specific may be accomplished and/or detected invitro or in vivo. In vitro binding, as described above, may be performedusing histologic specimens, or fractions or extracts of tissue or fluid,including substantially purified T cells or selected subsets of T cells,preferably CD4⁺ T cells. In vivo binding may be achieved byadministering the antibody (or fragment or derivative) by any route ormeans known in the art, including but not limited to intravenous,intraperitoneal, intranasal, and intraarterial, such that specificbinding may be detected. For detection of the TCR chain in cells in thelung, intrapulmonary administration, such as by inhalation of a spray ormist, may be used.

Imaging techniques can be used in vivo, wherein the antibody, derivativeor fragment is bound to a detectable label capable of in vivolocalization. Many different labels and methods of labeling are known inthe art.

The present invention provides method for diagnosing sarcoidosis basedon detecting the specific binding of a mAb, or a derivative or fragmentthereof, to T cells expressing the TCR V.sub.α 2.3 chain in a biologicalsample from a subject suspected of having the disease. Biologicalsamples which may be tested according to the present invention includeany body fluid, such as peripheral blood, plasma, cerebrospinal fluid,lymph, peritoneal fluid, or pleural fluid, and the like, or any bodytissue or extract thereof. Preferably samples for the diagnostic methodsof the present invention include blood, bronchoalveolar lavage fluid,lymph, lymph node tissue and lung tissue.

According to the present invention, sarcoidosis may be diagnosed in asubject by detecting the increased presence of T cells expressingV.sub.α 2.3, in particular CD4⁺ cells expressing V.sub.α 2.3, in abiological sample, such as bronchoalveolar lavage fluid, from thesubject as compared to a "baseline sample." As used herein, the term"baseline sample" refers to a sample from a normal, healthy individualwho does not have sarcoidosis (or who has a disease unrelated tosarcoidosis not known to involve any changes in the distribution ofpresence of V.sub.α 2.3⁺ cells) or a sample from the subject prior toonset of the disease or at a time of remission of the disease. Abaseline sample may also be a mixture or average of samples from ageneral population. In one embodiment, the biological sample beingtested is from the site of disease, generally lung tissue orbronchoalveolar lavage fluid, and the baseline sample is the peripheralblood.

Alternatively, such diagnosis may be achieved by detection of thepresence of nucleic acid sequences characteristic of the TCR V.sub.α 2.3regions using molecular techniques. Preferably, such molecular diagnosisis achieved by detecting the presence of nucleic acid sequenceshomologous to a gene encoding a part of the variable region of TCRV.sub.α 2.3 in mRNA in a patient sample. The nucleic acid sequence (SEQID NO:1) of TCR V.sub.α 2.3 DNA is shown in FIG. 4, and the amino acidsequence (SEQ ID NO:2) is shown in FIG. 5. One skilled in the art couldreadily design diagnostic tests to detect the presence of increased Tcells expressing V.sub.α 2.3 as described here. In one embodiment, mRNAencoding V.sub.α 2.3 in a sample is detected by Northern analysis, bycontacting an RNA-containing preparation with a nucleic acid probespecific for TCR V.sub.α 2.3 and detecting the hybridizationtherebetween. In another embodiment, DNA encoding V.sub.α 2.3 in asample is detected by Southern analysis, by contacting a DNA-containingsample with a nucleic acid probe specific for TCR V.sub.α 2.3 anddetecting the hybridization therebetween. Molecular approaches used tocorrelate TCR gene expression with disease include:

(1) producing and analyzing cDNA libraries obtained from thedisease-related T cells obtained from one or more subjects having thedisease, to determine the presence of frequently used or "dominant" TCRgenes;

(2) Southern analysis of disease samples to determine whether specificgenetic polymorphisms (e.g., RFLPs) or oligoclonal TCR rearrangementsexist;

(3) analysis of disease samples by cDNA synthesis, PCR amplification,and slot blot hybridization procedures;

(4) in situ nucleic acid hybridization of TCR probes to T cells withoutprior culture of these cells.

It should be understood that the diagnostic methods of the presentinvention are best used in conjunction with other known diagnosticmethods to obtain a comprehensive patient diagnosis. Although theprecise pathogenesis of sarcoidosis is not known, clinical evidencesuggests that the lung is the first site of involvement. The processextends through the lymphatics to the hilar and mediastinal lymph nodes.Radiographically apparent mediastinal and hilar lymph node involvementoccurs in about 90% of sarcoidosis patients. Thus, a diagnosis ofsarcoidosis; may be made based on the methods of the present inventiontogether with conventional diagnostic recognition of the clinicalfeatures of sarcoidosis, such as those described above. For a morecomplete description of clinical aspects of sarcoidosis, see, forexample, Neville, E. et al., Quart. J. Med. 208:525 (1983); Fanburg, B.L., In: Lung Biology in Health and Disease, C. Lefant, ed., M. DekkerInc., vol. 20 (1983); and Brostoff, J. et al., Clinical Immunology,Gower Medical Publishing (1991), which references are hereinincorporated by reference. As with any diagnostic criteria, theparameters disclosed in the present invention may not be soledeterminants, or pathognomonic, of sarcoidosis.

5.3. Therapeutic Use Antibodies of the Invention

As mentioned above, the present invention is also useful in the therapyof sarcoidosis. The therapeutic embodiments of the present inventionbased on the correlation between the disease and preferential use of theV.sub.α 2.3 gene in T cells associated with the disease or preferentialproliferation and/or accumulation of CD4⁺ V.sub.α 2.3⁺ T cells in thelungs of patients with sarcoidosis. The antibodies, fragments orderivatives of the present invention are therapeutically useful in partbecause they may interfere with the binding of the T cell, via its TCR,to the MHC/antigen complex needed for initiation or propagation of theinflammatory process underlying sarcoidosis.

T cells of the subset associated with sarcoidosis may recognize a true"sarcoidosis antigen" or an disease-associated antigen (such as certainviral or bacterial antigens). Such cells may be cloned and expanded orimmortalized in culture by methods well-known in the art. The culturedcells serve as the source of cell-surface TCR chains for making yetadditional antibodies or as the source of cDNA encoding the appropriateTCR for molecular identification of TCR usage. Such cDNA is cloned andexpressed by methods well known in the art. (See, for example, Sambrook,J. et al., (Molecular Cloning: A Laboratory Manual, 2nd Edition, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1989)).

Treatment of an individual according to this invention with antibodies,fragments or derivatives comprises parenterally administering a singledose or multiple doses of the antibody, fragment or derivative. Theeffective dose is a function of the individual antibody (or fragment orderivative), the presence and nature of a conjugated therapeutic agent,the subject and his clinical status. Effective doses of the antibodies,fragments or derivatives of this invention for use in preventing,suppressing, or treating an immune-related disease are in the range ofabout 1 ng to 100 mg/kg body weight. A preferred dose range is betweenabout 10 ng and 10 mg/kg. A more preferred dose range is between about100 ng and 1 mg/kg.

The route of administration may include intravenous (IV), subcutaneous(SC), intramuscular, intrapulmonary, intraperitoneal (IP), intranasal,intracerebroventricular, intrathecal, intradermal, or other knownroutes.

As mentioned above, the antibody or antigen-binding fragment thereof canbe coupled to cytotoxic proteins, including ribosomal inhibitoryproteins such as Ricin-A, Pseudomonas toxin, and Diphtheria toxin, aswell as other proteins such as tumor necrosis factor. Toxins conjugatedto antibodies or other ligands, are known in the art (see, for example,Olsnes, S. et al., Immunol. Today 10:291-295 (1989)). Since antibody toa particular TCR epitope will react with a much smaller proportion oftotal lymphocytes than the more broadly-reactive immunotoxins used todate, higher doses of a toxin-conjugated anti-TCR antibody will betolerated by patients, or conversely, lower doses will be effective.

In a preferred embodiment, ricin A chain is conjugated to a anti-V.sub.α2.3 antibody resulting in an immunoconjugate capable of binding to theTCR of lymphocytes which are a causative agent or pathophysiologicallysignificant agent of sarcoidosis and destroying the cells, therebytreating the disease. Effective doses of a ricin A conjugated monoclonalanti-TCR antibody are in the range of about 0.005 to 0.5 mg/kg/day, withthe preferred dose in the range of about 0.05 to 0.2 mg/kg/day.

The anti-TCR antibody, fragment or derivative can be conjugated toadditional types of therapeutic moieties including, but not limited to,radionuclides and cytotoxic drugs. Non-limiting examples ofradionuclides which can be coupled to antibodies and delivered in vivoto sites of antigen include ²¹² Bi, ¹³¹ I, ¹⁸⁶ Re, and ⁹⁰ Y. Suchradionuclides exert their cytotoxic effect by locally irradiating thecells, leading to various intracellular lesions, as is well-known in theart of radiotherapy.

Cytotoxic drugs which can be conjugated to antibodies and subsequentlyused for in vivo therapy include, but are not limited to, daunorubicn,doxorubicin, methotrexate, and mitomycin C. For a fuller exposition ofthese classes of drugs which are known in the art, and their mechanismsof action, see Goodman, A. G., et al., Goodman and Gilman's ThePharmacological Basis of Therapeutics, 7th Ed., Macmillan PublishingCo., (1985).

The therapeutic approaches disclosed herein are based on any of a numberof possible mechanisms by which the antibodies, fragments or derivativesof the present invention may act to achieve the therapeutic benefits.The present inventors do not intend to be bound by any particular theoryas to mechanism of action. For example, in one embodiment, an antibodydirected against TCR V.sub.α 2.3 can be used, alone or conjugated to atoxic agent, to remove the undesired T cells. In another embodiment, theantibody is administered therapeutically to block the interaction ofeffector T cells with the antigen for which they are specific, therebymodulating a deleterious immune response.

An administered antibody, fragment or derivative of the presentinvention may act by binding a V.sub.α 2.3 molecule in vivo and markingthe T cell bearing that TCR chain for elimination by the one or anotherhost defense system such as the reticuloendothelial system, or throughantibody-dependent cellular cytotoxicity.

For the antibody, fragment or derivative of the present invention to beuseful in therapy, it must have the ability to recognize and eithermodulate or lead to the destruction of the disease-related T cellsubset.

First generation treatments based on anti-TCR V region antibodytherapeutics may be developed using knowledge of the correlation betweensarcoidosis and the expression of a the TCR V.sub.α 2.3 gene subfamilyin subjects having the disease. Such therapeutics offer an improvementover current procedures. Prior to the present invention, therapeuticoptions for sarcoidosis included: (a) no therapy with possiblespontaneous resolution; (b) treatment with corticosteroids; (c)alternative forms of anti-inflammatory or immunosuppressive therapy,including nonsteroidal anti-inflammatory drugs, cyclosporin A,azathioprine, cyclophosphamide, chlorambucil, chloroquine, methotrexate,and calcium chelating agents. However, none of these drugs listed in (c)have clearly improved efficacy over corticosteroids. The therapeuticmethods of the present invention have the distinct advantage over theprior art methods of treating sarcoidosis in that the methods of thepresent invention target only the particular T cell subset expressing aparticular TCR Vα subfamily. These methods of the present invention moreclosely approach the goal of modulating only the disease-related Tcells, while sparing or not affecting other T cells in the subject, toachieve a greater specificity of therapy.

For measuring preventative, suppressive, or therapeutic benefit of theantibody (or TCR peptide; see below) compositions of this invention inhumans, certain clinical outcome measures are used. In sarcoidosis,clinical and laboratory parameters include: (a) clinical disability; (b)pulmonary function, (c) pulmonary radiography, e.g., presence ofperipheral and/or parenchymal lung lesions; (d) lymph node radiography,e.g., unilateral or bilateral enlargement; and (e) cytologicalexamination of bronchoalveolar lavage fluid, e.g., presence and numberof lymphocytes or T cell subsets.

5.4. T Cell Receptor-Based Peptides and Their Therapeutic Application

The present invention is also directed to treating sarcoidosis using apeptide corresponding to a portion of the TCR expressed on lymphocytesassociated with the disease, preferably the V.sub.α 2.3 chain. Thepresent inventors do not intend to be bound by the mechanism(s) by whichthe TCR protein or peptide acts on the immune system. According to oneembodiment, these TCR "mimic" peptides are able to interfere with thebinding of the MHC/antigen complex (or the antigen alone) needed forinitiation or propagation of the immunological stages of theinflammatory process of sarcoidosis. Alternatively or additionally, theTCR peptides stimulate the immune system to respond to the TCR on thedisease-mediating T cells, resulting in a therapeutic benefit associatedwith such "counter-autoimmunity."

In general, the TCR peptide sequence represents a portion of the TCRitself and preferably corresponds to a portion of the TCR which isextracellular, exposed to antibody or other T cells, and is ofbiological importance in the activity of the T cell bearing the TCR. Forthe purposes of this invention, the peptide is preferably immunogenic,that is, capable of inducing an immune response when injected into asubject. It is understood that the protein or peptide comprising the TCRpeptide according to this invention can be used alone or bound to, orcontained within the sequence of, a longer peptide. The longer peptidemay carry additional sequence derived from the V.sub.α 2.3 chain or mayinclude sequences of an unrelated peptide, such as a carrier proteinused to enhance the immunogenicity of the TCR oligopeptide. Suchcarriers are well known in the art and include heterologous proteinssuch as, for example, keyhole limpet hemocyanin (KLH), bovine serumalbumin, tetanus toxoid and the like. Also included within the scope ofthis invention are methods and compositions which utilize the TCRpeptide conjugated to an antibody or the peptide conjugated to a toxin.Toxins useful for such compositions and methods include the ribosomalinhibitory proteins, such as, for example, the ricin A chain,Pseudomonas toxin, Diphtheria toxin, as well as other proteins such astumor necrosis factor. Toxins conjugated to antibodies or other ligands,are known in the art (see, for example, Olsnes, S. et al., supra).

One peptide according to the present invention has 135 amino acids andincludes the leader sequence, and the V and J regions of V.sub.α 2.3.The amino acid sequence (in single letter code) of this peptide is:

MMISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFPNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNIRPGNTPLVFGKGTRLSVIPNI (SEQ ID NO:2, see FIG. 5).

A preferred peptide corresponds to the VJ region of SEQ ID NO:2,beginning at residue 24, and has the amino acid sequence:

KEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNIRPGNTPLVF GKGTRLSVIPNI (SEQ IDNO:3)

Alternatively, the peptide can correspond to the V region of SEQ IDNO:2, having the amino acid sequence:

KEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVN (SEQ ID NO:4.)

In another embodiment, the peptide corresponds to the J region of SEQ IDNO:2, having the amino acid sequence: IRPGNTPLVFGKGTRLSVIPNI (SEQ IDNO:5)

In a preferred embodiment, the peptide corresponds to at least part ofone of the three complementarity determining regions (CDRs) of the TCR αchain. The CDRs of the TCR are defined by analogy to the structure ofthe immunoglobulin molecule wherein CDRs comprise the amino acidsequences of the heavy or light chain V regions which contact theantigen and constitute crucial portions of the antigen-binding site. Allthree TCR CDRs are believed to participate in binding to antigen and MHC(Davis, M. M., et al.; Claverie, J. M., et al., Immunol. Today 10:10-14(1989)).

Thus, a peptide corresponding to CDR1 of the TCR V.sub.α 2.3 region is a34-mer with the sequence from residues 24-57 of SEQ ID NO:2:

KEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFW (SEQ ID NO:6)

A peptide corresponding to CDR2 of the TCR V.sub.α 2.3 region is a47-mer with the sequence from residues 58-104 of SEQ ID NO:2:

YRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSD (SEQ ID NO:7)

A peptide corresponding to CDR3 of the TCR V.sub.α 2.3 region is a31-mer with the sequence from residues 105-135, including both the V andJ region, of SEQ ID NO:2: SATYLCVVNIRPGNTPLVFGKGTRLSVIPNI (SEQ ID NO:8).

By directing the immune response of the subject to generate eitherprotective antibodies or regulatory T cells which are specific to one ofthe CDRs of the TCR V.sub.α 2.3 region, the likelihood of disruptingnecessary binding or recognition events between thesarcoidosis-associated T cell and the yet unknown autoantigen and/or theMHC is increased.

In a preferred aspect, the size of the peptide selected for use in thisinvention is sufficient for conferring antigenicity or immunogenicity,while maintaining the minimal epitope structure such that a T cell orantibody specific for the TCR peptide will recognize and react with theTCR V.sub.α 2.3 chain on an intact T cell. For example, peptides of thisinvention, in order to be sufficiently immunogenic and to have a highprobability of including the relevant epitope of the TCR which can leadto modulation of T cell activity, are of the range of at least about 6amino acids, more preferably at least about 15-30 amino acids, althoughpeptides of differing length are also contemplated. For example, a 21amino acid TCR peptide present on the TCR β chain associated withexperimental allergic encephalomyelitis (EAE) has been used to treat EAEsuccessfully (Vandenbark, A. A., et al., Nature 341:541-544 (1989);Vandenbark, A., PCT Publication WO 91/01133 (Feb. 7, 1991); see, alsoJaneway, C. A., Nature 341:482-483 (1989)). Others have reported thateven shorter peptides of 8 or 11 amino acids, corresponding to the βchain VDJ region or the Jα region proved effective as "vaccines" foreither preventing or reducing the severity of EAE in a rat model(Howell, M. D. et al., Science 246:668-671 (1989); Howell, M. D. et al.,PCT Publication WO 92/12996 (Aug. 6, 1992)).

By examining a variability plot of TCR α chains, such as that disclosedby Kabat, E. et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST,5th Edition, 1991, NIH, U.S. Dept. of Health and Human ServicesPublication #913242), one of ordinary skill in the art will discern theregions which possess the greatest variability, and from thisinformation, can determine the peptides which comprise 3 CDRs of the TCRα chain. Thus, preferred peptides for therapeutic uses in accordancewith the present invention include the following five peptides(designated P1-P5) corresponding to fragments of the TCR VJ α2.3 regionfrom SEQ ID NO:2:

      Residues    P1:      24-44           KEVEQDPGPFNVPEGATVAFN                                (SEQ ID NO:9)    P2:      45-61           CTYSNSASQSFFWYRQD    (SEQ ID NO:10)    P3:      62-77           CRKEPKLLMSVYSSGN     (SEQ ID NO:11)    P4:      78-109           EDGRFTAQLNRASQYISLLIRDSKLSDSATYL                                (SEQ ID NO:12)    P5:      110-135           CVVNIRPGNTPLVFGKGTRLSVIPNI                                (SEQ ID NO:13)

In light of the observations described above in the treatment of otherimmunological diseases associated with a particular TCR, therapeuticpeptides as short as about 15 amino acids from the α chain V or J regionmay be used to prevent the progression of sarcoidosis. It may also bepossible to prevent the onset of sarcoidosis by such treatment ifappropriately susceptible individuals, for example those expressingHLA-DR3(w17), can be identified. Identification of those individualshaving a particular HLA type can be performed using routine methodswell-known in the art. For detailed methods of serological andbiochemical analysis of HLA, see American Society for Histocompatibilityand Immunogenetics Laboratory Manual, 2nd Edition, Zachary, A. A. etal., eds., 1990 (herein incorporated by reference). For methods of DNAand molecular analysis of HLA, see, for example, Carlsson, B. et al.,Hum. Immunol. 20:95 (1987); Bidwell, J. L. et al., Baillieres Clin.Haematol. 3:355-384 (1990); Tiercy, J. M. et al., Blood Rev. 4:9-15(1990); Wordsworth, P., Immunol. Lett. 29:37-39 (1991); Shaffer, A. L.et al., Tiss. Antigens 39:84-90 (1992) (which references are hereinincorporated by reference).

Also intended within the scope of this invention is a "functionalderivative" of the TCR α chain peptide, including a "fragment,""variant," "analogue," or "chemical derivative" of the peptide, whichterms are defined below.

A "fragment" refers to any subset of the molecule, that is, a shorterpeptide. A "variant" of the peptide refers to a molecule substantiallysimilar to either the entire peptide or a fragment thereof. Variantpeptides may be conveniently prepared by direct chemical synthesis ofthe variant peptide, using methods well-known in the art. Alternatively,amino acid sequence variants of the peptide can be prepared by mutationsin the DNA which encodes the synthesized peptide. Such variants include,for example, deletions from, or insertions or substitutions of, residueswithin the amino acid sequence. Any combination of deletion, insertion,and substitution may also be made to arrive at the final construct,provided that the final construct possesses the desired activity.Obviously, the mutations that will be made in the DNA encoding thevariant peptide must not alter the reading frame and preferably will notcreate complementary regions that could produce secondary mRNA structure(see EPO Publication EP75444). At the genetic level, these variantsordinarily are prepared by site-directed mutagenesis of nucleotides inthe DNA encoding the peptide molecule, thereby producing DNA encodingthe variant, and thereafter expressing the DNA in recombinant cellculture. The variants typically exhibit the same qualitative biologicalactivity as the nonvariant peptide.

Another group of variants are those in which at least one amino acidresidue in the protein molecule, and preferably, only one, has beenremoved and a different residue inserted in its place. For a detaileddescription of protein chemistry and structure, see Schulz, G. E. etal., Principles of Protein Structure, Springer-Verlag, New York, 1978,and Creighton, T. E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated byreference. The types of substitutions which may be made in the TCRV.sub.α 2.3 a chain protein or peptide molecule may be based on analysisof the frequencies of amino acid changes between a homologous protein ofdifferent species, such as those presented in Table 1-2 of Schulz et al.(supra) and FIG. 3-9 of Creighton (supra). Based on such an analysis,conservative substitutions are defined herein as exchanges within one ofthe following five groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr(Pro, Gly);

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu,Gln;

3. Polar, positvely charged residues: His, Arg, Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and

5. Large aromatic residues: Phe, Tyr, Trp.

The three amino acid residues in parentheses above have special roles inprotein architecture. Glycine is the only residue lacking any side chainand thus imparts flexibility to the chain. Proline, because of itsunusual geometry, tightly constrains the chain. Cysteine can participatein disulfide bond formation which is important in protein folding.Tyrosine, because of its hydrogen bonding potential, has some kinshipwith serine and threonine.

Substantial changes in functional or immunological properties are madeby selecting substitutions that are less conservative, such as between,rather than within, the above five groups, which will differ moresignificantly in their effect on maintaining (a) the structure of thepeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Examplesof such substitutions are (a) substitution of glycine and/or proline byanother amino acid or deletion or insertion of glycine or proline; (b)substitution of a hydrophilic residue, e.g., serine or threoine, for (orby) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine,valine or alanine; (c) substitution of a cysteine residue for (or by)any other residue; (d) substitution of a residue having anelectropositive side chain, e.g., lysine, arginine or histidine, for (orby) a residue having an electronegative charge, e.g., glutamic acid oraspartic acid; or (e) substitution of a residue having a bulky sidechain, e.g., phenylalanine, for (or by) a residue not having such a sidechain, e.g., glycine.

The activity of a TCR protein or peptide variant is then screened in asuitable screening assay for the desired characteristic. For example, achange in the immunological character of the peptide molecule, such asbinding to a given anti-TCR mAb, is measured by a competitive typeimmunoassay. Changes in T cell recognition of the variant peptide ismeasured by a delayed hypersensitivity assay in vivo or a T cellproliferation assay in vitro, which are well-known in the art.Modifications of such peptide properties as redox or thermal stability,hydrophobicity, susceptibility to proteolytic degradation or thetendency to aggregate with carriers or into multimers are assayed bymethods well known to the ordinarily skilled artisan.

An "analog" of a peptide refers to a non-natural molecule substantiallysimilar to either the entire molecule or a fragment thereof.

A "chemical derivative" of a peptide of this invention containsadditional chemical moieties not normally a part of the peptide.Covalent modifications of the peptides are included within the scope ofthis invention. Such modifications may be introduced into the moleculeby reacting targeted amino acid residues of the peptide with an organicderivatizing agent that is capable of reacting with selected side chainsor terminal residues. Such derivatized moieties may improve thepeptide's solubility, absorption, biological half life, and the like.The moieties may alternatively eliminate or attenuate any undesirableside effect of the peptide and the like. Moieties capable of mediatingsuch effects are disclosed, for example, in Remington's PharmaceuticalSciences, 16th ed., Mack Publishing Co., Easton,

5.5. Antisense Oligonucleotides as Inhibitors of T Cells ExpressingV.sub.α 2.3

By the term "antisense" is intended an RNA sequence, as well as a DNAsequence coding therefor, which is sufficiently complementary to aparticular MRNA molecule ("sense" RNA) for which the antisense RNA isspecific to cause molecular hybridization between the antisense RNA andthe mRNA. The action of the antisense RNA results in specific inhibitionof gene expression in the cell (see: Albers, B. et al., MolecularBiology of the Cell, 2nd Ed., Garland Publishing, Inc., New York, N.Y.(1989), in particular, pages 195-196, which reference is hereinincorporated by reference).

An antisense nucleic acid of the present invention is preferably anoligonucleotide having at least about six nucleotides which can bedouble-stranded or single-stranded, RNA or DNA, or a modification of aderivative thereof, such as a nucleic acid containing nucleotide baseanalogues.

An oligonucleotide, between about 6 and about 100 bases in length,preferably at least about 15 nucleotides, and more preferably at leastabout 18 or about 25 nucleotides, and complementary to the targetsubsequence of the TCR V.sub.α 2.3 gene region (SEQ ID NO:1) may besynthesized by methods known in the art. For example, the antisenseoligonucleotide may be synthesized from natural mononucleosides or,alternatively, from mononucleosides having substitutions at thenon-bridging phosphorous bound oxygens (see below). In a pharmaceuticalcomposition useful for treating sarcoidosis, the oligonucleotide of thepresent invention is combined with a pharmaceutically acceptablecarrier.

Basic procedures for constructing recombinant DNA and RNA molecules inaccordance with the present invention are disclosed by Sambrook, J. etal., supra).

Oligonucleotide molecules having a strand which encodes antisense RNAcomplementary to the a TCR V.sub.α 2.3 sequence can be prepared usingprocedures which are well known to those of ordinary skill in the art(Belagaje, R., et al., J. Biol. Chem. 254:5765-5780 (1979); Maniatis,T., et al., In: Molecular Mechanisms in the Control of Gene Expression,Nierlich, D. P., et al., Eds., Acad. Press, NY (1976); Wu, R., et al.,Prog. Nucl. Acid Res. Molec. Biol. 21:101-141 (1978); Khorana, H. G.,Science 203:614-625 (1979)). Additionally, DNA synthesis may be achievedthrough the use of automated synthesizers. Techniques of nucleic acidhybridization are disclosed by Sambrook et al. (supra), and by Haymes,B. D., et al., In: Nucleic Acid Hybridization, A Practical Approach, IRLPress, Washington, D.C. (1985)), which references are hereinincorporated by reference.

An "expression vector" is a vector which (due to the presence ofappropriate transcriptional and/or translational control sequences) iscapable of expressing a DNA (or cDNA) molecule which has been clonedinto the vector and of thereby producing an RNA or protein product.Expression of the cloned sequences occurs when the expression vector isintroduced into an appropriate host cell. For expression of an antisenseDNA in a human T cell, a eukaryotic expression vector is employed.Preferred promoters and additional regulatory elements, such aspolyadenylation signals, are those which should yield maximum expressionin T lymphocytes. To efficiently express an antisense RNA complementaryto a TCR V.sub.α 2.3 sequence, a transcriptional control unit (promoterand polyadenylation signal) are selected which provide efficientexpression in lymphoid cells or tissues, in particular in human Tlymphocytes. Examples of useful viral and eukaryotic promoters includethe promoter of the mouse metallothionein I gene (Hamer, D., et al., J.Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus(McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter(Benoist, C. et al., Nature 290:304-310 (1981), all of which referencesare incorporated by reference herein). The most preferred promoters forexpression of antisense in T lymphocytes are the cytomegalovirusimmediate early promoter, optionally used in conjunction with the bovinegrowth hormone polyadenylation signals, and the promoter of theMoloney-MuLV LTR, for use with a lympholtropic retrovirus. Themetallothionein promoter has the advantage of inducibility.

A DNA sequence encoding the antisense RNA of the present invention maybe recombined with vector DNA in accordance with conventionaltechniques, including blunt-ended or staggered-ended termini forligation, restriction enzyme digestion to provide appropriate termini,filling in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and ligation with appropriateligases. Techniques for such manipulations are disclosed by Sambrook etal., supra, and are well known in the art.

According to the present invention, successful transfection of cellswith DNA antisense to the TCR V.sub.α 2.3 gene may inhibit thedevelopment of, or activity of, T cell bearing this TCR. Thus, growth,differentiation, activation or lung localization of V.sub.α 2.3-bearingT cells may be prevented or inhibited. This antisense DNA must havesufficient complementarity to the V.sub.α 2.3 gene so that the antisenseRNA can hybridize to the V.sub.α 2.3 DNA or mRNA and inhibit the gene'sexpression, regardless of whether the action is at the level ofsplicing, transcription or translation. Preferably, the oligonucleotidecomprises between about 15 and about 100 nucleotides complementary to apart of SEQ ID NO:1and is preferably at least 18 or at least 25nucleotides.

The antisense RNA of the present invention may be hybridizable to any ofseveral portions of the target V.sub.α 2.3 DNA, including the codingsequence, 3' or 5' untranslated regions, or other intronic sequences, orto V.sub.α 2.3 mRNA. As is readily discernible by one of ordinary skillin the art, the minimal amount of homology required by the presentinvention is that sufficient to result in hybridization to the V.sub.α2.3 DNA or mRNA and inhibition of transcription of the DNA ortranslation or function of the mRNA, while substantially not affectingthe function of other essential mRNA molecules and the expression ofother essential genes in the cells.

The antisense oligonucleotide of the present invention may include otherappending groups such as peptides, or agents facilitating transportacross cell membranes (Letsinger et al., Proc. Natl. Acad. Sci. USA86:6553-6556; Lemaitre et al., Proc. Natl. Acad. Sci. USA 84:648-652(1987); PCT Publication WO 88/09810 (Dec. 15, 1988)), hybridizationcleavage agents (Krol et al., BIoTechniques 6:958-976 (1988)) orintercalating agents (Zon et al., Pharm Res. 5:539-549 (1988)).

In one embodiment, antisense RNA is delivered to a cell bytransformation or transfection with a vector into which has been placedDNA encoding the antisense RNA with the appropriate regulatorysequences, including a promoter, which results in expression of theantisense RNA in a host cell. Means of delivery of such antisense RNA orantisense DNA are known (EPO Publication 248,531; PCT Publication WO89/2110; Lemaitre et al., Proc. Natl. Acad. Sci. U.S.A., 84:648-652(1987); (Toulme et al., Gene 72:51-58 (1988).

In another embodiment, the antisense oligonucleotide is directlydelivered to the cells, e.g., via receptor-mediated endocytosis, byformulation into a liposome which is taken up by cells, by directinjection into a cell, etc.

In addition to antisense oligonucleotides containing native nucleotides,the oligonucleotide of the present invention may include nucleoside ornucleotide analogues, having a modified sugar or modified phosphatebackbone. Such analogues have the advantageous properties of resistanceto nuclease hydrolysis and improved penetration into mammalian cells(Miller, P. S. et al., Biochemistry 20:1874-1880 (1981)). For example,an oligo(deoxyribonucleoside phosphonate) complementary to sequences ofviral, bacterial or eukaryotic DNA blocks gene expression (Jayaraman, K.et al., Proc. Natl. Acad. Sci. USA 78:1537-1541 (1983); Blake, K. R. etal., Biochemistry 24:6139-6145 (1985); Miller, P. et al., Feder. Proc.43:abstr. 1811 (1984); Smith, C. C. et al., Proc. Natl. Acad. Sci. USA83:2787-2791 (1986)). Preferred analogues which make an oligonucleotideresistant to in vivo degradation nucleases have modified internucleosidelinkages, for example, methylphosphonates, phosphorothioates, or2'-O-methylribose or 1'-alpha- anomers. More generally, preferredanalogues are mononucleoside analogues which result in anoligonucleotide which has improved diffusion through cell membranes orincreased resistance to nuclease digestion within the body of a subject.Such nucleoside analogues are well-known in the art, and their use inthe inhibition of gene expression are detailed in a number of references(Miller, P. S. et al., supra; Jayaraman, K. et al., supra; Blake, K. R.et al., supra; Smith, C. C. et al., supra). The entire antisenseoligonucleotide molecule may be formed of such modified linkages, oronly certain portions, such as the 5' and 3' ends, may be so affected,thereby providing resistance to exonucleases. Antisense moleculessuitable for use in the present invention include but are not limited todideoxyribonucleoside methylphosphonates (Mill, et al., Biochemistry18:5134-5143 (1979)), oligodeoxynucleotide phosphorothioates (Matsukuraet al., Proc. Nat. Acad. Sci., 84:7706-10 (1987)), oligodeoxynucleotidescovalently linked to an intercalating agent (Zerial et al., Nuc. AcidsRes. 15:9909-9919 (1987)), oligodeoxynucleotide conjugated withpoly-L-lysine (Leonetti et al., Gene 72:32-33 (1988), andcarbamate-linked oligomers assembled from ribose-derived subunits(Summerton, J., Antisense Nucleic Acids Conference 37:44 (1989)).

5.6. Pharamaceutical Compositions and Their Administration

The preclinical and clinical therapeutic use of the present invention inthe treatment of sarcoidosis will be best accomplished by those ofskill, employing accepted principles of diagnosis and treatment. Suchprinciples are known in the art, and are set forth, for example, inBraunwald, E. et al., eds., Harrison's Principles of Internal Medicine,11th Ed., McGraw- Hill, publisher, New York, N.Y. (1987).

The antibodies, fragments and derivatives of the present invention, andthe TCR V region peptides are well suited for the preparation ofpharmaceutical compositions. The pharmaceutical compositions of theinvention may be administered to any animal which may experience thebeneficial effects of the compositions of the invention. Foremost amongsuch animals are humans, although the invention is not intended to be solimited.

The pharmaceutical compositions of the present invention may beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intrapulmonary, intranasal, intradermal, intramuscular, intraperitoneal,transdermal, or buccal routes. Alternatively, or concurrently,administration may be by the oral route. The pharmaceutical compositionscan be administered parenterally by bolus injection or by gradualperfusion over time.

The dosage administered will be dependent upon the age, sex, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired. The dose ranges forthe administration of the compositions of the present invention arethose large enough to produce the desired effect. The doses should notbe so large as to cause adverse side effects, such as unwanted crossreactions, generalized immunosuppression, anaphylactic reactions and thelike.

Preferred doses for humans range between about 0.0001-25 mg of antibody,fragment or derivative per kg body weight. Preferred doses of the TCRpeptide for humans range between about 1-1000 mg per kg body weight.

In addition to the pharmacologically active components (i.e., antibody,fragment, or derivative, or TCR peptide), pharmaceutical compositionspreferably contain suitable pharmaceutically acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Such pharmaceutically acceptable carrier are sterile. Moreover, as usedherein, the term pharmaceutically acceptable carriers does not includecell culture media, or any components not approved for use in humans.

Suitable formulations for parenteral administration include aqueoussolutions of the antibody in water-soluble form, for example,water-soluble salts. In addition, suspensions of the antibody asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally,the suspension may also contain stabilizers. The antibodies, fragmentsor derivatives of the invention are preferably formulated in purifiedform substantially free of aggregates and other protein materials,preferably at concentrations of about 1.0 ng/ml to 100 mg/ml.

The compositions are formulated using conventional pharmaceuticallyacceptable parenteral vehicles for administration by injection. Thesevehicles are nontoxic and therapeutic, and a number of formulations areset forth in Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Co., Easton, Pa. (1980). Non-limiting examples of excipientsare water, saline, Ringer's solution, dextrose solution and Hank'sbalanced salt solution. Formulations according to the invention may alsocontain minor amounts of additives such as substances that maintainisotonicity, physiological pH, and stability.

To enhance delivery or bioactivity, the antibodies, fragment orderivative thereof, can be incorporated into liposomes using methods andcompounds known in the art.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

6. Example: Restricted V.sub.α 2.3 Gene Usage By CD4⁺ T Lymphocytes InBronchoalvelar Lavage Fluid From Sardoidosis Patients And CorrelationWith HLA-DR3 6.1. Materials and Methods 6.1.1. Subjects

The sarcoidosis population studied consisted of 11 patients withuntreated sarcoidosis (median age: 29-46); 2 women). one was a smoker(no. 9); eight were non-smokers and two ex-smokers (>5 years). Sixpatients had clinically active sarcoidosis (nos. 1, 2, 4, 5, 10 and 11).Patients nos. 5, 6, 7 and 10 were reanalyzed six months after theinitial investigation. During this period, patients nos. 6, 7 and 10were all untreated. When reanalyzing these patients, nos. 1, 7 and 10were clinically healthy. Patient no. 6 presented symptoms of coughing,low-grade fever and enlarged cervical lymph nodes (LN), one of which wasextirpated. Four healthy volunteers (median age 36 (20-59); 3 women),all non-smokers were included as controls.

6.1.2. Bronchoalveolar Lavage

Sterile saline, 250 ml at 37° C., was instilled in five aliquots of 50ml. The fluid was gently aspirated after each instillation and collectedin a siliconized bottle kept on ice. The mean recovery of the instilledfluid was 73±5% in the sarcoidosis patients and 75±5% in the controls.

6.1.3. Cells

The BAL fluid was strained through a double layer of Dacron nets. Cellswere pelleted by centrifugation at 400×g for 5 min at 4° C. andresuspended in Hanks balanced salt solution (HBSS). The median totalcell concentrations were 125±10⁶ (interquartile ranges (i.q.r.) 78-218)cells/l in samples from the investigated sarcoidosis patients, and68×10⁶ (45-93) cells/l from the controls. The median proportions ofmacrophages/monocytes, lymphocytes and neutrophils were 77% (64-87), 22%(12-34) and 0.8% (0.3-1.4) in the sarcoidosis patients and 91% (87-93)8% (6-10) and 1.0% (0.7-1.3) in the controls, respectively. Peripheralblood mononuclear cells (PBMC), separated from heparinized peripheralblood by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradientcentrifugation, were washed twice in RPMI-1640 medium (Gibco, Paisley,Scotland) and diluted in PBS.

LN were extirpated under sterile conditions, rinsed thoroughly in RPMImedium, minced into small sections with sterile scissors and pressedthrough sterile stainless steel mesh. Cells were washed twice in RPMImedium, minced into small sections with sterile scissors and pressedthrough sterile stainless steel mesh. Cells were washed twice in RPMImedium and lymphocytes were separated by Ficoll-Hypaque gradientcentrifugation.

6.1.4. Monoclonal Antibodies

The following mAbs specific for TCR V gene segments were tested:

    ______________________________________    Specificity               mAb      Reference    ______________________________________    V.sub.β 5.1               LC4      Maecker et al., J. Immunol. 142:                        1395 (1989)    V.sub.β 5.2 + 5.3               1Cl      Boylston et al., J. Immunol. 137:                        741 (1989)    V.sub.β 5.3               W112     Tian et al., Proc. J. 3:A486 (1989)    V.sub.β 6.7               OT145    Posnett et al., Proc. Natl. Acad.                        Sci. 83:7888 (1986)    V.sub.β 8               16G8     Tian et al., supra    V.sub.β 12               S511     Bigler et al., J. Exp. Med. 158:                        1000 (1983)    V.sub.α 12.1               6D6      DerSimonian et al., J. Exp. Med.                        174:639 (1991)    V.sub.α 2.3               F1       Janson et al., Cancer Immunol.                        Immunother 28:225 (1989), produced                        in the present inventors' lab    ______________________________________

The reactivities of these mAb may include more V gene members thanindicated above.

The OKT3 (anti-CD3) hybridoma was acquired from American Type CultureCollection (Rockville, Md.). Phycoerythrin (PE)-conjugated mAbs Leu-3a(anti-CD4), IL2R (anti-CD25), HLA-DR and Leu-18 (anti CD45) wereobtained from Becton Dickinson (Mountain View, Calif.).

The mAb 4B4 (anti-CD29) was purchased from Coulter Corporation, Inc.(Hialeah, Fla.).

The TCR δ1 mAb (T Cell Sciences Inc.) was used as a marker for the γ/δTCR.

FITC-conjugated F(ab')₂ fragments of rabbit anti-mouse Ig were obtainedfrom Dakopatts A/S (Glostrup, Denmark). Normal mouse serum (NMS,produced from BALB/c mice, was used for negative control at a dilutionof 1:500 in PBS.

6.1.5. Flow Cytometric Analysis

Cells were incubated for 30 min with unlabeled TCR-specific mAb or NMS,washed twice with PBS and incubated with FITC-conjugated F(ab')₂fragments of rabbit anti-mouse Ig for 30 min. The cells were washedthree times with PBS. NMS, diluted 1:500, was thereafter added to blockremaining rabbit anti-mouse Ig. After 10 min., PE-conjugated mAb wasadded, the cells were incubated for 20 min and washed twice in PBS. Tenthousand cells were analyzed in a FACScan® flow cytometer (BectonDickinson) and a Hewlett Packard 300 computer (Palo Alto, Calif.).Lymphocytes were gated out by forward and side scatter, and dead cellsby staining with propidium iodide. NMS, used as negative control alwaysstained ≦0.5% of the cell population. Optimal compensation was set forgreen and orange fluorescence.

6.1.6. Definition of T Cell Subpopulations

The CD4⁺ T subpopulation was defined as those cells which were Leu-3⁺,OKT3⁺, and TcRδ1⁻. An abnormal compartmentalization was characterized asmore than >10% CD4⁺ T in BAL occurring together with a more than 3-foldincreased reactivity for a particular TCR-specific mAb in BAL ascompared to PBL

6.1.7. HLA Typing

HLA class I antigens (A, B and C) and DR antigens were determined by themicrolymphocytotoxicity technique well-known in the art using antiserawhich were available locally or commercially. Genomic HLA-DR and HLA-DQtyping was performed using TaqI RFLP analysis (Carlsson, B. et al.,supra).

6.1.8. Statistical Analysis

The differential counts of alveolar cells and the percentages of cellspositive in a reaction with a given mAb are presented as the medianswith i.q.r. The p values were obtained by the use of the non-parametricWilcoxon-Mann-Whitney two-tailed test.

6.2. Results 6.2.1. Characterization of T Lymphocytes from PBL and BAL

In line with earlier findings, the present study showed an accumulationof Th cells (CD4⁺) in the alveolar space of sarcoidosis patients (Table1). The median percentage of CD4⁺ T cells in the total T cell populationwas 84% in BAL versus 57% in PBL.

The accumulated BAL CD3⁺ cells showed signs of activation, unlike thePBL T cells. Thus, 40% of the CD3⁺ cells in BAL expressed HLA-DR,compared to 5.4% in PBL. The expression of CD25 did not show such adifference: 3.0% of CD3⁺ BAL cells were CD25⁺ and 2.6% of CD3⁺ PBL wereCD25⁺.

The CD29 molecule is expressed by memory cells (Sanders, M. E. et al.,J. Immunol. 140:1401 (1988)), and the CD4⁺ T cells which are also CD29⁺have been defined as a "helper inducer" subset (Morimoto, C. et al., J.Immunol. 134:3762 (1985)). The median percentage of CD29⁺ cells in theCD4⁺ BAL cell population was 96%. In contrast, only 51% of CD4⁺ PBL wereCD29⁺.

The mAb Leu-18 (CD45RA) defines the "suppressor inducer" subset of Tcells (Morimoto, C. et al., J. Immunol. 134:1508 (1985)) which includes"virgin" cells (Kristensson, K. et al., Scand. J. Imunol. 32:243(1990)). The expression of CD45RA⁺ cells in sarcoidosis patients alsomarkedly differed between the two compartments: about 1% of CD4⁺ BALcells were CD45RA⁺ in contrast to 43% of CD4⁺ PBLs.

                  TABLE 1    ______________________________________    Characterization of PBL and BAL T Cells    from Sarcoidosis Patients nos. 1-11.sup.a    Subset         PBL          BAL    ______________________________________    γδ.sup.+ /CD3.sup.+                   6.7 (4.2-12.5)                                1.8 (1.0-2.4)    IL2R.sup.+ /CD3.sup.+                   2.6 (1.9-5.0)                                3.0 (2.4-4.6)    HLA-DR.sup.+ /CD3.sup.+                   5.4 (4.3-5.8)                                40.0 (22-49)    CD4.sup.+ /CD3.sup.+                   57.1 (47-73) 83.8 (70-87)    CD29.sup.+ /CD4.sup.+b                   51.0 (28-73) 96.0 (93-98)    CD45RA.sup.+ /CD4.sup.+c                   43.0 (36-55) 0.6 (0.4-1.0)    ______________________________________     .sup.a Bold figures show the median reactivity, as percent of CD3.sup.+     or CD4.sup.+  cells within PBL and BAL. Underlined values are abnormal.     Figures in parentheses show the i.q.r. (P25-P75).     .sup.b Normal value is 41% (Morimoto et al., J. Immunol. 134:3762 (1985))     .sup.c Normal values are 41% (Morimoto et al., J. Immunol. 134:1508     (1985)).

6.2.2. TCR V Gene Expression in CD4⁺ Cells

The reactivity of the TCR-specific mAbs with BAL cells and PBL werecompared. In 27% (3/11) of the sarcoidosis patients, distinct signs ofcompartmentalization of CD4⁺ T lymphocytes in the lung were evident(FIG. 1, Table 2).

In all three of these patients, the localized CD4⁺ BAL T cells werereactive with the V.sub.α 2.3-specific mAb. In patient no. 10, 31.9% ofthe CD4⁺ BAL cells were V.sub.α 2.3⁺ (FIG. 2), compared to 3.8% in PBL.In patients nos. 6 and 7, 21.7% and 19.6%, respectively, of BAL CD4⁺ Tcells were V.sub.α 2.3⁺ and 6.8% and 4.5%, respectively, of CD4⁺ PBLwere V.sub.α 2.3⁺. Additionally, patient no. 1 showed an accumulation ofV.sub.α 2.3⁺ cells among CD4⁺ T cells in the lung, with a more than3-fold increase in percentage (9.0%) compared to PBL (2.8%).

In the initial study, no correlation was observed between thedistribution of V.sub.α 2.3⁺ CD4⁺ T cells in the lung and active vs.inactive disease, acute vs. non-acute disease, or chest radiography.However, subsequent analyses of larger number of patients showed arelationship between lung localization and sarcoidosis (see below). Noother such lung compartmentalization was observed in cells reacting withany of the other TCR α-specific mAbs (Table 2).

The four patients showing compartmentalization of V.sub.α 2.3⁺ CD4⁺ Tcells to the lung (No's. 1, 6, 7 and 10) were subjected to an additionalround of reanalysis (Table 3, FIG. 3) 6 months, or later, after theinitial test. During this period, patients nos. 1 and 10 appeared tohave been clinically cured, either spontaneously (No. 10) or bysteroid-treatment (No. 1). Patients No. 6 and 7 did not present with anysubjective symptoms at the time of initial investigation, and patientNo. 7 appeared well at the time of the first and second analysis.

At the time of reanalysis, Patient No. 6 had symptoms of activesarcoidosis disease including coughing, fever and enlarged cervical LN.

As can be seen in FIG. 3 and Table 3, after cure, patients No. 1 and 10had normalized values of V.sub.α 2.3⁺ CD4⁺ T cells in BAL, whiledisplaying virtually the same normal frequencies of V.sub.α 2.3⁺ CD4⁺ Tcells in PBL.

In contrast, patient no. 6, in particular, but also patient no. 7,maintained the abnormal compartmentalization of such T cells in BAL.1

                                      TABLE 2    __________________________________________________________________________    Percent of CD4.sup.+  BAL Lymphocytes or CD4.sup.+  PBL Reactive with TCR    Specific mAb    in Sarcoidosis Patients (1-11) and Healthy Controls (A-D)    TCR-specific mAb    Subject        V.sub.α 2.3             V.sub.α 12.1                 V.sub.β 5.1                      V.sub.β 5.2 + 5.3                            V.sub.β 5.3                                V.sub.β 6.7                                    V.sub.β 8                                        V.sub.β 12    __________________________________________________________________________    1   9.0/2.8             0.9/4.4                 4.0/4.4                      3.4/2.3                            1.3/0.8                                5.6/9.8                                    6.7/3.6                                        1.7/1.1    2   4.5/3.3             3.4/3.4                 4.5/5.9                      3.4/3.0                            1.9/1.0                                6.1/3.6                                    3.2/5.8                                        2.0/2.1    3   2.0/2.9             2.1/4.4                 4.7/4.9                      2.6/2.8                            0.8/1.2                                5.1/3.7                                    3.0/3.3                                        1.0/1.6    4   3.4/ND             3.1/ND                 10.2/ND                      1.4/ND                            0.9/ND                                3.7/ND                                    3.2/ND                                        1.2/ND    5   3.0/3.9             1.3/3.1                 6.3/5.3                      3.4/3.0                            2.2/1.0                                7.1/5.9                                    7.9/5.6                                        1.3/1.4    6   21.7/6.8.sup.a             1.5/2.2                 4.6/5.6                      5.3/2.9                            2.0/1.3                                8.2/5.5                                    3.3/2.9                                        3.0/1.9    7   19.6/4.5             1.6/2.3                 4.4/4.9                      2.1/2.5                            0.8/0.9                                6.5/3.8                                    2.8/4.2                                        0.7/1.1    8   4.1/3.5             3.3/3.3                 2.8/4.7                      3.9/2.1                            2.6/0.6                                4.0/3.5                                    4.9/4.0                                        1.5/1.5    9   4.8/4.8             2.4/2.8                 3.5/4.6                      3.5/2.1                            1.6/1.2                                5.1/3.6                                    5.2/9.1                                        0.8/2.5    10  31.9/3.8             0.8/2.8                 5.7/6.3                      4.0/2.1                            2.2/1.0                                6.8/6.7                                    5.8/4.3                                        1.9/2.1    11  1.8/3.2             3.6/2.4                 5.1/4.0                      3.5/3.4                            1.3/1.4                                4.7/3.7                                    8.2/4.5                                        2.1/1.8    A.sup.b)        6.0/3.3             3.1/3.1                 3.3/3.5                      3.7/2.0                            0.8/0.9                                6.2/3.2                                    3.1/4.2                                        4.4/2.0    B   3.0/4.1             ND/ND                 3.7/3.7                      1.8/2.2                            0.5/0.9                                6.8/4.3                                    9.1/4.1                                        8.4/1.6    C   7.6/5.1             1.4/1.7                 7.6/7.4                      5.5/2.5                            ND/ND                                1.4/2.3                                    3.9/4.6                                        2.4/2.0    D   7.8/2.7             ND/1.6                 ND/5.4                      ND/3.1                            ND/0.8                                ND/5.2                                    ND/4.9                                        ND/2.0    __________________________________________________________________________     .sup.a Underlined figures in table show signs of compartmentalization of     TCR.sup.+  cells (i.e. greater than three times the value of percentage i     BAL compared to PBL, plus >10% reactivity in BAL) to the lung.     .sup.b A-D are healthy controls. A and B are HLADR3.sup.- ; C and D are     HLADR3.sup.+.

                                      TABLE 3    __________________________________________________________________________    Second Analysis of Sarcoidosis Patient Lymphocyte Reactivity with    Anti-TcR mAb    Anti-TcR mAb    Patients         V.sub.α 2.3              V.sub.α 12.1                   V.sub.β 5.1                       V.sub.β 5.2 + 5.3                             V.sub.β 6.7                                  V.sub.β 8                                      V.sub.β 12    __________________________________________________________________________    1    4.5/2.9.sup.1              ND/3.9                   ND/3.4                       ND/2.5                             ND/6.7                                  6.3/2.6                                      ND/1.2    6    20.6/6.6.sup.2              1.3/2.4                   3.8/4.9                       5.5/3.8                             9.7/4.9                                  2.9/3.3                                      2.8/2.6    7    12.2/3.6              ND/2.1                   ND/5.3                       ND/3.8                             ND/3.8                                  ND/4.2                                      ND/1.6    10   6.8/4.6              1.8/2.8                   3.5/4.5                       8.3/5.6                             8.3/5.6                                  2.9/4.4                                      1.5/2.5    6/LN.sup.3         7.0  2.8  4.6 3.0   7.0  4.1 2.3    __________________________________________________________________________     .sup.1 % of CD4.sup.+  BAL/% of CD4.sup.+  PBL reactive with mAb     .sup.2 Underlined figures show signs of compartmentalization of TCR.sup.+     cells.     .sup.3 6/LN represents cervical lymph node lymphocytes from patient 6.

Analysis of total cell and lymphocyte counts in BAL in these fourpatients revealed a decrease in the total number of lymphocytes inpatients No. 1 and 10, in contrast to patients No. 6 and 7 (Table 4). Inaddition, the number of CD3⁺ CD4⁺ cells was reduced in BAL, but not inPBL, in patients 1 and 10, as compared to patients 6 and 7 who still hadhigh percentages of CD3⁺ CD4⁺ cells in BAL (Table 5).

Patient 6 presented with enlarged cervical LN at the time of the secondanalysis. One such LN was extirpated and analyzed for TCR V gene usage.Interestingly, the percent of cells expressing the V.sub.α 2.3 gene wassimilar in the LN (7.0%) and the PBL (6.6%) populations (Table 3).

                  TABLE 4    ______________________________________    Total Cell and Lymphocyte Counts in BAL of Sarcoidosis    Patients at Time of Disease Onset (First Analysis)    and More than 6 Months Later (Second Analysis)            First analysis    Second analysis    Patient TCC.sup.a  LC.sup.b TCC     LC    ______________________________________    1       144        25 (18%).sup.c                                84      11 (14%)    6       125        28 (22%) 95      33 (34%)    7        60         5 (9%)  94       9 (9%)    10      152        61 (40%) 101      5 (5%)    ______________________________________     .sup.a TCC = Total cell count (× 10.sup.6 /l).     .sup.b LC =Lymphocyte count (× 10.sup.6 /l).     .sup.c Numbers in parentheses are the percentage of lymphocytes in the     total cell count.

                  TABLE 5    ______________________________________    Percentages of CD3.sup.+  and CD4.sup.+  BAL Lymphocytes in    Sarcoidosis Patients at Time of Disease Onset (First    Analysis) and More than 6 Months Later (Second Analysis)           First analysis    Second analysis    Patient  % CD3.sup.+                     % CD4.sup.+ % CD3.sup.+                                       % CD4.sup.+    ______________________________________    1        72      46          22    15    6        82      74          88    82    7        50      35          89    62    10       97      93          92    64    ______________________________________

6.2.3. HLA Typing

Since the TCR recognize antigen in the context of MHC, it was importantto define the HLA phenotypes of the patients, to understand the possiblerelation between selective TCR usage in compartmentalized T cells ofsarcoidosis patients with MHC antigen expression in these cellpopulations. Thus, patients and controls were HLA typed (Table 6).

Interestingly, the three patients with highly significantcompartmentalization of V.sub.α 2.3⁺ CD4⁺ T cells in BAL (No. 6, 7 and10) were all HLA-B8, Cw7, DR3(w17), DQw2⁺. DRw17 is a recentlyintroduced split of DR3. A fourth patient (no. 1) with a moderateaccumulation of V.sub.α 2.3⁺ CD4⁺ T cells in BAL also expressed theHLA-DR3(w17), DQw2 haplotype.

                                      TABLE 6    __________________________________________________________________________    HLA Haplotypes of Sarcoidosis Patients and Healthy Controls    HLA-    Patient         A     B    C      DR       DQ    __________________________________________________________________________    1.sup.a         A2    B7,15                    Cw3,w7?                           DR2(w15), 3(w17).sup.b                                    DQw1(w6),w2    2    A2,3  B7,15                    Cw3,w7 DR2(w15),4                                    DQW1(w6),w3(w8)    3    A2,3  B7,18                    Cw7    DR2(w15),5(w11)                                    DQw1(w6),w3(w7)    4    A9(24),11               B5,12(44)                    Cw8?   DR4,7    DQw2,w3(w8)    5    A2    B5,13                    Cw2?,w5?,w6?                           DR2(w15), 5(w11)                                    DQw1(w6),w3(w7)    6    A3,w19(31)               B7,8 CW7    DR2(w15), 3(w17)                                    DQw1(w6),w2    7    A1,11 B8,27                    Cw2,w7?                           DR3(w17),4                                    DQw2, w3(w8)    8    A3,w19(32)               B12,27                    Cwl,w5 DR1,2(w15)                                    DQw1(w5),w1(w6)    9    A9(24)               B5,40                    Cw3?   DR2(w15),4                                    DQw1(w6),w3(w8)    10   A1, w19(29)               B8,12w                    Cw6,w7 DR3(w17),4                                    DQw2,w3(w7)    11   ND.sup.c               ND   ND     DRw6,w14 DQw1,ws    Control.sup.d    A    A2    B12(44)                    Cw5    DR1, 2   ND    B    ND    ND   ND     DR2(w16),7                                    DQw3(w7),w3(w9)    C    A1, 3 B8,27                    Cw2,w7?                           DR1,3    ND    D    A1, 2 B8,27                    Cw2,w7?                           DR2,3    ND    __________________________________________________________________________     .sup.a Patients whose identification numbers are underlined show     compartmentalization or accumulation of V.sub.α 2.3.sup.+ CD4.sup.+     cells in the lung.     .sup.b Underlined HLA types are HLADR3(w17) and/or DQW/2 haplotypes.     .sup.c ND = not determined     .sup.d A-D refer to healthy controls. A and B are HLADR3.sup.-, C and D     are HLADR3.sup.+.

In contrast, all remaining patients were DR3(w17), DQw2⁻.

The positive correlation found between expression of the HLA-DR3(w17),DQw2 haplotype and the ratio of V.sub.α 2.3 positivity between CD4⁺ BALand CD4⁺ PBL was highly significant (p<0.002). No statisticallysignificant correlations were observed between the HLA-DR3(w17), DQw2haplotype and the BAL/PBL, ratio of cells expressing any other TCR chainbased on reactivity with other anti-TCR mAbs.

6.2.4. Controls

No compartmentalization of any of the TCR mAb-reactive cells wasdetected in any of four healthy controls (Table 2). Two of theseindividuals were HLA-DR3⁺.

6.2.5. Analysis of Additional Patients

Studies performed subsequent to those described above analyzed the CD4⁺T lymphocytes in BAL and PBL of sarcoidosis patients that were either ofthe HLA-DR3(w17) type (designated DR3⁺) or were of other HLA-DR types(designated DR3⁻). A total of 27 patients (inclusive of those describedabove) were studied, 12 DR3⁺ and 15 DR3⁻. The results of expression ofTCR α or β chains in the PBL or BAL of these patients, or of controls,compared by HLA-DR type or by PBL vs. BAL, are summarized in Tables 7-11below.

                  TABLE 7    ______________________________________    PERCENTAGE OF CD4.sup.+  T LYMPHOCYTES EXPRESSING VARIOUS    TCR α or β CHAINS IN SARCOIDOSIS PATIENTS (n = 27)           LYMPHOCYTES FROM:             Bronchoalveolar                          Peripheral    TCR      Lavage       Blood        p-value    ______________________________________    Vα2.3             4.5 (3.4-14.8)                          3.4 (3.0-4.3)                                       0.015*    Vα12.1             1.6 (1.1-2.4)                          2.7 (2.2-3.3)                                       0.001*    Vβ2 11.0 (7.4-13.8)                          9.8 (9.0-11.1)                                       --    Vβ3 2.7 (1.4-5.0)                          5.2 (1.6-5.4)                                       --    Vβ5.1             4.6 (3.6-5.7)                          4.9 (4.6-6.1)                                       0.242    Vβ6 5.1 (3.6-6.3)                          3.8 (3.4-5.0)                                       0.226    Vβ8 4.1 (3.1-5.5)                          4.4 (3.8-5.5)                                       0.516    Vβ12             1.9 (1.2-2.5)                          1.8 (1.4-2.1)                                       0.582    ______________________________________     Results represent the median and confidence interval (10%-90% of the     percentage of cells staining as positive with mAbs which characterize the     denoted TCR chain.     *statistically significant difference between BAL lymphocytes and PRL     (Student's t test)

                  TABLE 8    ______________________________________    PERCENTAGE OF CD4.sup.+  T LYMPHOCYTES EXPRESSING VARIOUS    TCR α or β CHAINS IN HLA-DR3-POSITIVE    SARCOIDOSIS PATIENTS (n = 12)           LYMPHOCYTES FROM:             Bronchoalveolar                          Peripheral    TCR      Lavage       Blood        p-value    ______________________________________    Vα2.3             17.5 (11.9-27.4)                          4.1 (3.6-5.2)                                       <0.001*    Vα12.1             0.9 (0.8-1.7)                          2.4 (2.1-2.9)                                       <0.001*    Vβ2 8.4          10.7 (9.4-13.4)                                       --    Vβ3 5.9          3.7 (1.7-5.4)                                       --    Vβ5.1             4.6 (4.2-5.7)                          6.0 (5.1-6.4)                                       0.159    Vβ6 5.6 (2.8-6.7)                          4.1 (3.9-5.3)                                       0.390    Vβ8 3.9 (3.1-5.3)                          4.3 (4.1-4.7)                                       0.435    Vβ12             2.3 (1.8-3.8)                          1.9 (1.2-2.0)                                       0.121    ______________________________________     Results represent the median and confidence interval (10%-90% of the     percentage of cells staining as positive with mAbs which characterize the     denoted TCR chain.     *statistically significant difference between BAL lymphocytes and PRL     (Student's t test)

                  TABLE 9    ______________________________________    PERCENTAGE OF CD4.sup.+  T LYMPHOCYTES EXPRESSING VARIOUS    TCR α or β CHAINS IN HLA-DR3-NEGATIVE    SARCOIDOSIS PATIENTS (n = 15)           LYMPHOCYTES FROM:             Bronchoalveolar                          Peripheral    TCR      Lavage       Blood        p-value    ______________________________________    Vα2.3             3.5 (2.6-4.1)                          3.1 (2.7-3.3)                                       0.303    Vα12.1             2.1 (1.4-3.2)                          3.0 (2.4-3.4)                                       0.075    Vβ2 12.9 (9.2-15.5)                          9.1 (7.8-9.7)                                       --    Vβ3 2.7 (1.1-3.2)                          5.2 (1.9-5.3)                                       --    Vβ5.1             4.6 (3.0-5.8)                          4.8 (4.3-5.3)                                       0.697    Vβ6 4.5 (3.6-5.6)                          3.6 (3.1-3.8)                                       0.226    Vβ8 4.5 (3.1-5.4)                          4.6 (3.4-5.7)                                       0.645    Vβ12             1.4 (1.1-2.2)                          1.7 (1.4-2.2)                                       0.472    ______________________________________     Results represent the median and confidence interval (10%-90% of the     percentage of cells staining as positive with mAbs which characterize the     denoted TCR chain.     *statistically significant difference between BAL lymphocytes and PRL     (Student's t test)

                  TABLE 10    ______________________________________    COMPARISON OF CD4.sup.+  T LYMPHOCYTES EXPRESSING VARIOUS    TCR α or β CHAINS IN BRONCHOALVEOLAR LAVAGE FLUID    LYMPHOCYTES IN HLA-DR3-POSITIVE VERSUS HLA-DR3-    NEGATIVE SARCOIDOSIS PATIENTS    TCR     HLA-DR3.sup.+ HLA-DR3.sup.- p-value5)    ______________________________________    Vα2.3            17.5 (11.9-27.4)                          3.5 (2.6-4.1) <0.001*    Vα12.1            0.9 (0.8-4.7) 2.1 (1.4-3.2) 0.013*    Vβ2            8.4           12.9 (9.2-15.5)                                        --    Vβ3            5.9           2.7 (1.1-3.2) --    Vβ5.1            4.6 (4.2-5.7) 4.6 (3.0-5.8) 0.555    Vβ6            5.6 (2.8-6.7) 4.5 (3.6-5.6) 0.238    Vβ8            3.9 (3.1-5.3) 4.5 (3.1-5.4) 0.818    Vβ12            2.3 (1.8-3.8) 1.4 (1.1-2.2) 0.150    ______________________________________     Results represent the median and confidence interval (10%-90% of the     percentage of cells staining as positive with mAbs which characterize the     denoted TCR chain.     *statistically significant difference between BAL lymphocytes from     DR3.sup.+  patients (n = 12) versus DR3.sup.-  patient (n = 15) (Student'     t test)

                  TABLE 11    ______________________________________    COMPARISON OF CD4.sup.+  T LYMPHOCYTES EXPRESSING VARIOUS    TCR α or β CHAINS IN PERIPHERAL BLOOD OF    HLA-DR3-POSITIVE VERSUS HLA-DR3-NEGATIVE    SARCOIDOSIS PATIENTS    TCR     HLA-DR3.sup.+ HLA-DR3.sup.- p-value5)    ______________________________________    Vα2.3            4.1 (3.6-5.2) 3.1 (2.7-3.3) 0.004*    Vα12.1            2.4 (2.1-2.9) 3.0 (2.4-3.4) 0.134    Vβ2            10.7 (9.4-13.4)                          9.1 (7.8-9.7)    Vβ3            3.7 (1.7-5.4) 5.2 (1.9-5.3)    Vβ5.1            6.0 (5.1-6.4) 4.8 (4.3-5.3) 0.020*    Vβ6            4.1 (3.9-5.3) 3.6 (3.1-3.8) 0.075    Vβ8            4.3 (4.1-4.7) 4.6 (3.4-5.7) 0.803    Vβ12            1.9 (1.2-2.0) 1.7 (1.4-2.2) 0.741    ______________________________________     Results represent the median and confidence interval (10%-90% of the     percentage of cells staining as positive with mAbs which characterize the     denoted TCR chain.     *statistically significant difference between PBL from DR3.sup.+  patient     (n = 12) versus DR3.sup.-  patient (n = 15) (Student's t test)

6.3. Discussion

The HLA-DR3(w17) antigen, expressed by 17.2% of the Swedish population,is associated with autoimmune diseases such as SLE, juvenile diabetes,Graves'disease and celiac disease (Tiwari, J. et al., HLA and DiseaseAssociation (1985)). Thus, in a subgroup of sarcoidosis patients, thepresent inventors have identified two of the structures in the specifictrimolecular complex constituted by the TCR, MHC and antigen. Thisfinding suggests the presence of a specific antigen being responsiblefor the pulmonary disease in sarcoidosis patients, here expressed asV.sub.α 2.3⁺ CD4⁺ TCR linked to the HLA-DR3(w17), DQW2 haplotype.

The references cited above are all incorporated by reference herein,whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains.

7. Deposits

One illustrative hybridoma cell line designated TM 19 MCB secretingmonoclonal antibody F1 was deposited on Nov. 4, 1992, at the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.,20110-2209,and was given accession number ATCC #HB 11176.

The methods and compositions of the present invention are not intendedto be limited to this cell line or its monoclonal antibody products.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 13    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 406 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..406    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - ATG ATG ATA TCC TTG AGA GTT TTA CTG GTG AT - #C CTG TGG CTT CAG TTA      48    Met Met Ile Ser Leu Arg Val Leu Leu Val Il - #e Leu Trp Leu Gln Leu    #                 15    - AGC TGG GTT TGG AGC CAA CGG AAG GAG GTG GA - #G CAG GAT CCT GGA CCC      96    Ser Trp Val Trp Ser Gln Arg Lys Glu Val Gl - #u Gln Asp Pro Gly Pro    #             30    - TTC AAT GTT CCA GAG GGA GCC ACT GTC GCT TT - #C AAC TGT ACT TAC AGC     144    Phe Asn Val Pro Glu Gly Ala Thr Val Ala Ph - #e Asn Cys Thr Tyr Ser    #         45    - AAC AGT GCT TCT CAG TCT TTC TTC TGG TAC AG - #A CAG GAT TGC AGG AAA     192    Asn Ser Ala Ser Gln Ser Phe Phe Trp Tyr Ar - #g Gln Asp Cys Arg Lys    #     60    - GAA CCT AAG TTG CTG ATG TCC GTA TAC TCC AG - #T GGT AAT GAA GAT GGA     240    Glu Pro Lys Leu Leu Met Ser Val Tyr Ser Se - #r Gly Asn Glu Asp Gly    # 80    - AGG TTT ACA GCA CAG CTC AAT AGA GCC AGC CA - #G TAT ATT TCC CTG CTC     288    Arg Phe Thr Ala Gln Leu Asn Arg Ala Ser Gl - #n Tyr Ile Ser Leu Leu    #                 95    - ATC AGA GAC TCC AAG CTC AGT GAT TCA GCC AC - #C TAC CTC TGT GTG GTG     336    Ile Arg Asp Ser Lys Leu Ser Asp Ser Ala Th - #r Tyr Leu Cys Val Val    #           110    - AAC ATT CGC CCA GGA AAC ACA CCT CTT GTC TT - #T GGA AAG GGC ACA AGA     384    Asn Ile Arg Pro Gly Asn Thr Pro Leu Val Ph - #e Gly Lys Gly Thr Arg    #       125    #                406A AAT ATC  C    Leu Ser Val Ile Pro Asn Ile    #   135    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 135 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Met Met Ile Ser Leu Arg Val Leu Leu Val Il - #e Leu Trp Leu Gln Leu    #                 15    - Ser Trp Val Trp Ser Gln Arg Lys Glu Val Gl - #u Gln Asp Pro Gly Pro    #             30    - Phe Asn Val Pro Glu Gly Ala Thr Val Ala Ph - #e Asn Cys Thr Tyr Ser    #         45    - Asn Ser Ala Ser Gln Ser Phe Phe Trp Tyr Ar - #g Gln Asp Cys Arg Lys    #     60    - Glu Pro Lys Leu Leu Met Ser Val Tyr Ser Se - #r Gly Asn Glu Asp Gly    # 80    - Arg Phe Thr Ala Gln Leu Asn Arg Ala Ser Gl - #n Tyr Ile Ser Leu Leu    #                 95    - Ile Arg Asp Ser Lys Leu Ser Asp Ser Ala Th - #r Tyr Leu Cys Val Val    #           110    - Asn Ile Arg Pro Gly Asn Thr Pro Leu Val Ph - #e Gly Lys Gly Thr Arg    #       125    - Leu Ser Val Ile Pro Asn Ile    #   135    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 112 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1...112    #/label= Name OTHER INFORMATION:    #"Corresponding to VJ region of SEQ ID NO:2"    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - Lys Glu Val Glu Gln Asp Pro Gly Pro Phe As - #n Val Pro Glu Gly Ala    #                15    - Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn Se - #r Ala Ser Gln Ser Phe    #            30    - Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu Pr - #o Lys Leu Leu Met Ser    #        45    - Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg Ph - #e Thr Ala Gln Leu Asn    #    60    - Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile Ar - #g Asp Ser Lys Leu Ser    #80    - Asp Ser Ala Thr Tyr Leu Cys Val Val Asn Il - #e Arg Pro Gly Asn Thr    #                95    - Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Se - #r Val Ile Pro Asn Ile    #           110    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 90 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1..90    #/label= Name OTHER INFORMATION:    #"Corresponding to V region of SEQ ID NO:2"    #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ    - Lys Glu Val Glu Gln Asp Pro Gly Pro Phe As - #n Val Pro Glu Gly Ala    #                15    - Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn Se - #r Ala Ser Gln Ser Phe    #            30    - Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu Pr - #o Lys Leu Leu Met Ser    #        45    - Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg Ph - #e Thr Ala Gln Leu Asn    #    60    - Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile Ar - #g Asp Ser Lys Leu Ser    #80    - Asp Ser Ala Thr Tyr Leu Cys Val Val Asn    #                90    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 22 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1..22    #/note= "Corresponding to J region    #ID 2"         of SEQ    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - Ile Arg Pro Gly Asn Thr Pro Leu Val Phe Gl - #y Lys Gly Thr Arg Leu    #                15    - Ser Val Ile Pro Asn Ile                20    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 34 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1..34    #/note= "Corresponding to CDR1 of    #V 2.3 region" the TCR    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - Lys Glu Val Glu Gln Asp Pro Gly Pro Phe As - #n Val Pro Glu Gly Ala    #                15    - Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn Se - #r Ala Ser Gln Ser Phe    #            30    - Phe Trp    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1..47    #/note= "Corresponding to CDR2 of    #V 2.3 region."the TCR    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - Tyr Arg Gln Asp Cys Arg Lys Glu Pro Lys Le - #u Leu Met Ser Val Tyr    #                15    - Ser Ser Gly Asn Glu Asp Gly Arg Phe Thr Al - #a Gln Leu Asn Arg Ala    #            30    - Ser Gln Tyr Ile Ser Leu Leu Ile Arg Asp Se - #r Lys Leu Ser Asp    #        45    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 31 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1...31    #/note= "Corresponding to CDR3 of the    #2.3 region"   TCR V    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    - Ser Ala Thr Tyr Leu Cys Val Val Asn Ile Ar - #g Pro Gly Asn Thr Pro    #                15    - Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Va - #l Ile Pro Asn Ile    #            30    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 21 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1..21    #/label= P1D) OTHER INFORMATION:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - Lys Glu Val Glu Gln Asp Pro Gly Pro Phe As - #n Val Pro Glu Gly Ala    #                15    - Thr Val Ala Phe Asn                20    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 17 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1..17    #/label= P2D) OTHER INFORMATION:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    - Cys Thr Tyr Ser Asn Ser Ala Ser Gln Ser Ph - #e Phe Trp Tyr Arg Gln    #                15    - Asp    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 16 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1..16    #/label= P3D) OTHER INFORMATION:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    - Cys Arg Lys Glu Pro Lys Leu Leu Met Ser Va - #l Tyr Ser Ser Gly Asn    #                15    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 32 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1..32    #/label= P4D) OTHER INFORMATION:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    - Glu Asp Gly Arg Phe Thr Ala Gln Leu Asn Ar - #g Ala Ser Gln Tyr Ile    #                15    - Ser Leu Leu Ile Arg Asp Ser Lys Leu Ser As - #p Ser Ala Thr Tyr Leu    #            30    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 26 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1..26    #/label= P5D) OTHER INFORMATION:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    - Cys Val Val Asn Ile Arg Pro Gly Asn Thr Pr - #o Leu Val Phe Gly Lys    #                15    - Gly Thr Arg Leu Ser Val Ile Pro Asn Ile    #            25    __________________________________________________________________________

What is claimed is:
 1. A method of treating sarcoidosis in a subject,comprising administering to the subject an amount effective fortreatment of sarcoidosis of a monoclonal antibody specific for anepiitope of the variable region of the T cell receptor V.sub.α 2.3chain, or an antigen-binding fragment or derivative of the monoclonalantibody.
 2. A method according to claim 1 wherein the monoclonalantibody, fragment, or derivative is linked to a pharmacologic agent. 3.A method according to claim 1, wherein the monoclonal antibody is F1, asproduced by the hybridoma deposited with the ATCC and assigned accessionnumber HB 11176.